WO1998017683A2 - Human phosphatase inhibitor-1 gene and methods of screening for non-insulin dependent diabetes mellitus - Google Patents

Abstract

An isolated DNA encoding human protein phosphatase inhibitor-1 (PPI-1) is disclosed. Methods for screening subjects for an increased risk of non-insulin dependent diabetes mellitus (NIDDM), based upon the detection of mutated PPI-1 or a DNA encoding mutated PPI-1 are also disclosed.

Description

HUMAN PHOSPHATASE INHIBITOR-1 GENE

AND METHODS OF SCREENING FOR NON-INSULIN

DEPENDENT DIABETES MELLITUS

This invention was made with Government support under Grant No. RO3-DK41190 from the National Institutes of Health. The Government has certain rights to this invention.

Field of the Invention

The present invention relates to the identification of DNA encoding the human protein phosphatase inhibitor-1 (PPI-1) and the use thereof in screening for non-insulin dependent diabetes mellitus

(NIDDM) .

Background of the Invention

Reversible protein phosphorylation controls many processes in plant and animal cells. P. Cohen, Proc. R. Soc . Lond. (Biol . ) 234, 115-144 (1988). As physiological responses of cells to hormones and other extracellular stimuli are defined by the phosphorylation state of key proteins, coordinating the opposing actions of protein kinases and phosphatases becomes important for cells to evoke an appropriate hormonal response. For example, Huang and Glinsmann {Eur. J. Biochem . 70, 419-426 (1976)) identified a protein from rabbit skeletal muscle extracts that, when phosphorylated by cyclic AMP-dependent protein kinase (PKA) , inhibited protein phosphatase activity. The phosphoprotein, inhibitor-1 (I-l), provided a potential mechanism for communication between a protein kinase and phosphatase that amplified hormonal signals mediated by the second messenger, cAMP. Purified I-l has also been purified from rabbit skeletal muscle. Using this purified I-l, it has also been shown that following PKA phosphorylation, nanomolar concentrations of I-l inhibited protein phosphatase-1 PP1) . G. Nimmo and P. Cohen, Eur. J. Biochem. 87, 341-351 (1978) . By comparison, concentrations of dephosphorylated I-l approaching 1 μM did not inhibit PP1 activity.

Hormones that elevate intracellular cAMP have been shown to increase I-l activity in many tissues. See, e.g., B. Khatra et al., FEES Lett . 114, 253-256 (1980); J. Foulkes & P. Cohen, Eur. J. Biochem . 97, 251-256 (1981); R. Nemenoff et al . , J. Biol Chem . 258, 9437-9443 (1983). As PP1 dephosphorylates numerous phosphoproteins, I-l activation may impose cAMP control over proteins that are not directly phosphorylated by PKA. Following a rise in intracellular calcium, I-l is inactivated by a calcium/calmodulin-activated protein phosphatase, known as calcineurin or PP2B, resulting in increased PP1 activity.

Such a cascade of phosphatases has been shown to control synaptic plasticity in hippocampal neurons. R. Mulkey et al., Nature 369, 486-488 (1994).

Inhibition by phosphorylated I-l and the structurally unrelated inhibitor-2 (1-2) has become a cornerstone of the classification scheme that characterizes protein serine/threonine phosphatases in mammalian tissues and many organisms. See T. Ingebritsen & P. Cohen, Science 221, 331-338 (1983); T. Ingebritsen et al., Eur. J. Biochem . 132, 297-307 (1983); P. Cohen, Annu . Rev. Biochem . 58, 453-508 (1989); S. Shenolikar & A. Nairn, Adv. Second Messengers Phosphoprotein Res . 23, 1- 121 (1991) . Type-1 phosphatases (PPl) are inhibited by I- 1 and 1-2. In contrast, several hundred-fold higher concentrations of these proteins have been shown to have no effect on type-2 phosphatases. G. Nimmo & P. Cohen, Eur. J. Biochem . 87, 353-365 (1978) . I-l appears to be an excellent substrate for the type-2 phosphatases, PP2A and PP2B, whose catalytic subunits share some structural homology with PP1. However, the molecular basis underlying I-l's function as a PP1 inhibitor and a substrate for PP2A and PP2B remains poorly understood.

Activation of glycogen synthase and glycogen synthesis in response to insulin is regulated partly through dephosphorylation by protein phosphatase-1 (PP1) , and partly through the insulin-stimulated inhibition of glycogen synthase kinase (GSK-2). Protein phosphatase inhibitor (PPI-1) is a potent inhibitor of PP1 activity when phosphorylated by cAMP-dependent protein kinase in response to epinephrine. This phenomenon provides molecular evidence for this hormone's antagonist effect to insulin. An increased activity of PPI-1 should inhibit PPl activity and decrease glycogen synthesis. The gene encoding PPI-1 is thus a good candidate gene useful for the screening for inherited insulin resistant glycogen synthesis seen in non-insulin dependent diabetic (NIDDM) patients, and their l^st-degree relatives. It would be desirable to be able to utilize this gene, and mutations thereof, in methods for screening populations for the risk of developing NIDDM.

Summary of the Invention

A first aspect of the present invention is an isolated DNA encoding the human protein phosphatase inhibitor-1 (PPI-1) .

A second aspect of the present invention is a method of detecting DNA encoding human protein phosphatase inhibitor-1 (PPI-1) in a sample DNA, comprising contacting an oligonucleotide probe that specifically binds to DNA encoding PPI-1 to the sample DNA, and then detecting the presence or absence of binding of the oligonucleotide probe to the sample DNA, with the presence of binding indicating the presence of DNA encoding human PPI-1 in the sample.

A third aspect of the present invention is a method for screening a subject for increased risk of non- insulin dependent diabetes mellitus (NIDDM) , comprising detecting the presence or absence of an inactivating mutation in the protein phosphatase inhibitor-1 (PPI-1) gene in the subject, and observing whether or not the subject is at increased risk of NIDDM by observing if a PPI-1 mutation is or is not detected, wherein the presence of a PPI-1 mutation indicates that the subject is at increased risk for NIDDM.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth hereinbelow.

Brief Description of the Drawings

Figure 1. Purification of Reco binant Human Inhibitor-1. E . coli (BL21) were transformed with plasmid pGEX-2T-hI-l and GST-I-1 expression was induced with IPTG. Figure 1A shows an SDS-PAGE of total bacterial extract (40 μg of total protein) in lane 1; lane 2 - GST- I-1 (1 μg) after affinity chromatography on glutathione- Sepharose; lane 3 - GST-I-1 (1 μg) after preparative SDS- PAGE. Phosphorylase b (M_r 97,400), bovine serum albumin (M_r 66,200), ovalbumin (M^r 45,000), carbonic anhydrase (M_r 31,000), soybean /trypsin inhibitor (M_r 21,500), and lysozyme (M_r 14,400) were used as molecular weight markers, and proteins were stained with Coomassie blue.

Figure IB shows an autoradiogram of GST-I-1 phosphorylated with PKA and [γ-³²P] ATP-Mg. Figure 2. Purification of Wild-type and Mutant

Inhibitor-1. Wild-type GST-I-1 and GST-I-1 (T35A) were purified from bacterial extracts by affinity chromatography on glutathione-Sepharose. The fusion proteins were digested with thrombin and wild-type and mutant I-l was purified on preparative SDS-PAGE. Figure 2A shows SDS-PAGE of the fusion proteins, wild-type GST-I-1 (lane 1), GST-I-1 (T35A) (lane 2) and I- 1 wild-type (lane 3) and mutant (lane 4) obtained from thrombin digestion of the fusion proteins. Lane 5 shows rabbit skeletal muscle I-l. Proteins were stained with Coomassie Blue.

Figure 2B shows an immunoblot of recombinant human I-l proteins with a polyclonal antibody generated against rabbit I-l. Lane 1: wild-type GST-I-1; lane 2: GST-I-1 (T35A) ; lane 3: wild-type I-l; lane 4: I-1(T35A); lane 5: rabbit skeletal muscle I-l . Phosphorylase b (M_r 97,400), bovine serum albumin (M_r 66,200), ovalbumin (M r 45,000), carbonic anhydrase (M_r 31,000), soybean trypsin inhibitor (M_r 21,500), and lysozyme (M_r 14,400) were used as molecular weight markers. Figure 3. Characterization of Inhibitor-1

Peptides. GST-I-1 was expressed in E. coli JM109 at 37°C. Figure 3A shows an SDS-PAGE of two I-l peptides obtained by thrombin cleavage of the 37 kDa fusion protein (lane 1) . Peptide a (lane 2) and b (lane 3) were individually purified on preparative SDS-PAGE and reversed-phase HPLC. To determine their C-termini, each peptide was digested with endoproteinase Glu-C and subjected to affinity chromatography on anhydrotrypsin-Sepharose. Peptides eluted from this affinity matrix were further purified by reversed-phase HPLC (Figure 3B) . A single major peptide (marked by arrow) was obtained from peptide a and peptide b .

Figure 3C shows N- and C-terminal sequences (in capital letters) obtained from each I-l peptide. The molecular weight of the peptides calculated from their amino acid sequence matches their mass as determined by mass spectrometry. Figure 4. N-terminal Truncation Inactivates An Inhibitor-1 Peptide. A synthetic gene encoding 1-1(9-54) was expressed in E. coli N4830 as fusion protein with Staphylococcus aureus protein A. The fusion protein was purified on IgG-Sepharose, digested with Factor Xa and re- chromatographed on IgG-Sepharose to yield 1-1(9-54) peptide. Parallel incubations (60 min at 37°C) containing the peptide (25 μg) and the catalytic subunit of PKA (5 μg) were carried out with (solid circles) and without 1 mM ATP and 10 mM MgCl2 (open circles) . Reactions were terminated by heating at 95°C for 10 min and unphosphorylated (open circles) and phosphorylated (solid circles) 1-1(9-54) was assayed for PP1 inhibition (Left Panel). EcoRI-Kpnl digestion excised a short 5 '-sequence to generate a synthetic gene that expressed protein A fused 1-1(13-54). The fusion protein was expressed, purified and digested with Factor Xa to yield 1-1(13-54). The peptide was incubated with PKA in the presence (solid circles) and absence of ATP-Mg (open circles) and subsequently assayed for PP1 inhibition (Right Panel) . Alignment of amino acid sequences for 1-1(9-54) and I- 1(13-54) with regions of homology between I-l and DARPP-32 is shown. Threonine phosphorylated by PKA is indicated by the arrowhead. Additional amino acids derived from the linker are underlined.

Detailed Description of the Invention

Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC- IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR Dl.822 and established usage. See, e.g., Patentln User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office) .

A. DNA sequences

DNAs of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:l and encoding the protein given herein as SEQ ID NO:2. This definition is intended to encompass natural allelic variations therein. Thus, isolated DNA or cloned genes of the present invention can be of any species of origin, including mouse, rat, rabbit, cat, porcine, and human, but are preferably of mammalian origin. Thus, DNAs which hybridize to DNA disclosed herein as SEQ ID NO:l (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention (e.g., a protein according to SEQ ID NO: 2) are also an aspect of this invention. Conditions which will permit other DNAs which code on expression for a protein of the present invention to hybridize to the DNA of SEQ ID NO:l disclosed herein can be determined in accordance with known techniques. For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt ' s solution, 0.5% SDS and lx SSPE at 37 °C; conditions represented by a wash stringency of 40-45% Formamide with 5x Denhardt ' s solution, 0.5% SDS, and lx SSPE at 42°C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt ' s solution, 0.5% SDS and lx SSPE at 42°C, respectively) to DNA of SEQ ID NO:l disclosed herein in a standard hybridization assay. See , e . g. , J. Sambrook et al., Molecular Cloning, A Labora tory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory) ) . In general, sequences which code for proteins of the present invention and which hybridize to the DNA of SEQ ID NO:l disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:l. Further, DNAs which code for proteins of the present invention, or DNAs which hybridize to that of SEQ ID NO:l, but which differ in codon sequence from SEQ ID NO:l due to the degeneracy of the genetic code, are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e . g. , U.S. Patent No. 4,757,006 to Toole et al. at Col. 2, Table 1 (applicant specifically intends that the disclosures of all U.S. patent references disclosed herein be incorporated herein by reference) .

B. Genetic Engineering Techniques The production of cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering is well known. See, e . g. , U.S. Patent No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Patent No. 4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Patent No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Patent No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59.

A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding the proteins of the present invention or to express the proteins of the present invention. An expression vector is a replicable DNA construct in which a DNA sequence encoding the proteins of the present invention is operably linked to suitable control sequences capable of effecting the expression of proteins of the present invention in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants .

Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus) , phage, retroviruses and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination) . The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself.

Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism.

DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase.

Transformed host cells are cells which have been transformed or transfected with vectors containing DNA coding for proteins of the present invention, constructed using recombinant DNA techniques. Transformed host cells ordinarily express protein, but host cells transformed for purposes of cloning or amplifying DNA coding for the proteins of the present invention need not express protein .

Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic organism cells. Prokaryote host cells include gram negative or gram positive organisms, for example, Escherichia coli (E. coli ) or Bacilli . Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using the plasmid pBR322. See Bolivar et al . , Gene 2, 95 (1977) .

Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host.

Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase

(penicillinase) and lactose promoter systems (Chang et al., Na ture 275, 615 (1978); and Goeddel et al., Na ture

281, 544 (1979)), a tryptophan (trp) promoter system

(Goeddel et al., Nucleic Acids Res . 8, 4057 (1980) and EPO

App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc . Na tl . Acad. Sci . USA 80, 21 (1983)). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the protein in plasmid or viral vectors (Siebenlist et al., Cell 20, 269 (1980)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA of the present invention, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA.

Eukaryotic microbes such as yeast cultures may be transformed with suitable protein-encoding vectors.

See, e . g. , U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Na ture 282, 39 (1979); Kingsman et al., Gene 1 , 141 (1979); Tschemper et al., Gene 10, 157 (1980)). This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1

(Jones, Genetics 85, 12 (1977)). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phospho- glycerate kinase (Hitzeman et al., J. Biol . Chem . 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)), such as enolase, glyceral- dehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657. Cultures of cells derived from multicellular organisms are a desirable host for recombinant protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect cells. Propagation of such cells in cell culture has become a routine procedure.

See Tissue Cul ture, Academic Press, Kruse and Patterson, editors (1973) . Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40) . See, e . g. , U.S. Patent No. 4,599,308. The early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al., Nature 273, 113 (1978). Further, the protein promoter, control and/or signal sequences, may also be used, provided such control sequences are compatible with the host cell chosen.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV) , or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to make proteins useful in carrying out the present invention, as described in U.S. Patents Nos . 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the chimeric protein DNA. An example of a suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. See U.S. Pat. No. 4,399,216. Such markers are proteins, generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein.

C . Methods of Screening for NIDDM As noted above, the present invention provides a method of screening (e.g., diagnosing or prognosing) for non-insulin dependent diabetes mellitus (NIDDM) in a subject. The method comprises detecting the presence or absence of a mutated PPI-1, or of DNA encoding a mutated PPI-1 in the subject. The presence of PPI-1 containing an inactivating mutation, or DNA containing such a mutation indicates that the subject is afflicted with NIDDM or at risk of developing NIDDM. Suitable subjects include those who have not previously been diagnosed as afflicted with NIDDM, those who have previously been determined to be at risk of developing NIDDM, and those who have been initially diagnosed as being afflicted with NIDDM where confirming information is desired. For example, patients diagnosed or determined to be afflicted with NIDDM, particularly patients who had previously been clinically normal and who are determined to be afflicted with NIDDM, are suitable subjects. Thus, the present invention may be employed in detecting both familial NIDDM (late onset and early onset) as well as sporadic NIDDM. Many NIDDM patients encountered in practice have no obvious family history and have been classified as sporadic. However, genetic factors in early- and late-onset familial NIDDM (FAD) are well documented. Late-onset NIDDM is the classification typically used if the disease is diagnosed as occurring after the age of 65 in humans.

Observing whether or not an inactivating mutation of PPI-1 is present or absent in a subject enables one to observe or determine whether or not a subject is afflicted with or at increased risk of developing NIDDM. Affliction with the disease is more likely if such a mutation is present. A subject with DNA containing an inactivating mutation of PPI-1 is at increased risk of developing NIDDM over subjects in which such a mutation is absent. A subject who is "at increased risk of developing NIDDM" is one who is predisposed to the disease, has genetic susceptibility for the disease or is more likely to develop the disease than subjects in which the PPI-1 mutation is absent.

The method of the present invention may be used to detect the presence of absence of any detectable mutation in either the protein PPI-1 or the gene encoding PPI-1. Mutations that may be detected by the invention include frame-shift mutations, deletion mutations (wherein an amino acid or nucleotide base present in the wild type protein or DNA is absent) , and substitution mutations, wherein an amino acid (of the peptide) or a nucleotide base (of the DNA) present in the wild-type of the protein or the DNA is replaced with another amino acid or nucleic acid. Additionally, mutations may be silent (for example, a nucleotide base substitution that does not affect the protein coding sequence) or not. For example, the method of the present invention may be used to detect the presence of a frame-shift mutation in codon 10 of the gene encoding PPI-1. This mutation occurs in a small subset of NIDDM patients (approximately less than 7%) . Another mutation that may be detected by the method of the present mutation includes a substitution mutation at the threonine-35 position of PPI-1, in which a the phosphoacceptor site at threonine-35 is mutated, generally by replacing threonine with a non-phosphorylated residue such as alanine. Yet another mutation that may be detected by the method of the present invention is a mutation in the DNA encoding PPI-1 in which the ^ΛA' at base position 435 is replaced with a G' . This mutation is silent mutation which was found after scanning the coding region of the DNA encoding PPI-1 by RT-PCR-SSCP/Heteroduplex analysis on skeletal muscle cDNA of NIDDM patients. The mutation has been found to be present in approximately 28 percent of individuals with NIDDM, but to date has not been detected in normal individuals. These examples of mutations that may be screened by the method of the present invention are provided as illustration only, in that the method is similarly useful in detecting other mutations that effect the likelihood of a subject developing NIDDM.

Further, the methods of the present invention can be used to aid in determining the prognosis of a subject afflicted with or at risk for NIDDM based on the observation of how many alleles containing a PPI-1 mutation are detected in the subject. The subject's prognosis is more negative if the presence of a mutation of PPI-1 is detected than if it is absent; the subject's prognosis is most negative if the presence of more than one allele for a mutated PPI-1 is detected. Further, the average age of onset of NIDDM and the average age of survival is younger for those having one mutated PPI-1 allele, and youngest for those having two mutated PPI-1 alleles. Thus, a subject's prognosis for NIDDM is more likely to be negative if the subject has a mutated PPI-1 allele and most negative if the subject has more than one mutated PPI-1 allele. The negative prognosis can be viewed in terms of increased likelihood of developing the disease, or of increased likelihood of developing the disease or dying at an earlier age.

It is preferred and contemplated that the methods described herein be used in conjunction with other clinical diagnostic information known or described in the art which are used in evaluation of subjects with NIDDM or suspected to be at risk for developing such disease.

D. Methods of Detecting Inactivating Mutations in PPI-1

The step of detecting the presence or absence of mutated PPI-2 or of DNA encoding such a mutation

(including the number of alleles for mutated PPI-1) may be carried out either directly or indirectly by any suitable means. A variety of techniques are known to those skilled in the art. All generally involve the step of collecting a sample of biological material containing either DNA or PPI-1 from the subject, and then detecting whether or not the subject possesses a mutation of PPI-1 or DNA encoding such a mutation from that sample. For example, the detecting step may be carried out by collecting an PPI-1 sample from the subject (for example, from blood, or any other fluid or tissue containing PPI-1) , and then determining the presence or absence of a mutation in the PPI-1 sample (e.g., by isoelectric focusing or immunoassay) .

In the alternative, the detecting step may be carried out by collecting a biological sample containing DNA from the subject, and then determining the presence or absence of DNA encoding PPI-1 containing an inactivating mutation in the biological sample. Any biological sample which contains the DNA of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source. Determining the presence or absence of DNA encoding a PPI-1 mutation may be carried out with an oligonucleotide probe labelled with a suitable detectable group, or by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction (the product of which amplification reaction may then be detected with a labelled oligonucleotide probe or a number of other techniques) . Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the gene encoding the mutated PPI-1. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention. See, e . g. , U.S. Patent No. 4,302,204 to Wahl et al . ; U.S. Patent No. 4,358,535 to Falkow et al . ; U.S. Patent No. 4,563,419 to Ranki et al . ; and U.S. Patent No. 4,994,373 to Stavrianopoulos et al .

Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means . See generally D. Kwoh and T. Kwoh, Am. Biotechnol . Lab . 8, 14-25 (1990) . Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification (see generally G. Walker et al . , Proc . Na tl . Acad. Sci . USA 89, 392-396 (1992); G. Walker et al . , Nucleic Acids Res . 20, 1691-1696 (1992)), transcription-based amplification (see D. Kwoh et al . , Proc . Na tl . Acad Sci . USA 86, 1173-1177 (1989)), self- sustained sequence replication (or "3SR") (see J. Guatelli et al . , Proc . Na tl . Acad. Sci . USA 87, 1874-1878 (1990)), the Qβ replicase system (see P. Lizardi et al . , BioTechnology 6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or "NASBA") (see R. Lewis, Genetic Engineering News 12(9), 1 (1992)), the repair chain reaction (or "RCR") (see R. Lewis, supra) , and boomerang DNA amplification (or "BDA") (see R. Lewis, supra ) . Polymerase chain reaction is currently preferred.

DNA amplification techniques such as the foregoing can involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding a mutated PPI-1, but do not bind to wild-type DNA encoding normal PPI-1 under the same hybridization conditions, and which serve as the primer or primers for the amplification of the mutated DNA or a portion thereof in the amplification reaction (likewise, one may use a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding wild-type PPI-1, but do not bind to DNA encoding mutated PPI-1 under the same hybridization conditions, and which serve as the primer or primers for the amplification of the normal DNA or a portion thereof in the amplification reaction.

In general, an oligonucleotide probe which is used to detect DNA encoding a mutated PPI-1 is an oligonucleotide probe which binds to DNA encoding mutated PPI-1, but does not bind to DNA encoding normal PPI-1 under the same hybridization conditions. The oligonucleotide probe is labelled with a suitable detectable group, such as those set forth below in connection with antibodies. Likewise, an oligonucleotide probe which is used to detect DNA encoding wild-type PPI-1 is an oligonucleotide probe which binds to DNA encoding wild-type PPI-1 but does not bind to DNA encoding mutated PPI-1 under the same hybridization conditions.

Polymerase chain reaction (PCR) may be carried out in accordance with known techniques. See, e . g. , U.S. Patents Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present. These steps are cyclically repeated until the desired degree of amplification is obtained. Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g., an oligonucleotide probe of the present invention) , the probe carrying a detectable label, and then detecting the label in accordance with known techniques, or by direct visualization on a gel. When PCR conditions allow for amplification of all PPI-1 types, the types can be distinguished by hybridization with allelic specific probe, by restriction endonuclease digestion, by electrophoresis on denaturing gradient gels, or other techniques .

Ligase chain reaction (LCR) is also carried out in accordance with known techniques. See, e . g. , R. Weiss, Science 254, 1292 (1991) . In general, the reaction is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. Each pair together completely overlaps the strand to which it corresponds. The reaction is carried out by, first, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes is ligated together, then separating the reaction product, and then cyclically repeating the process until the sequence has been amplified to the desired degree. Detection may then be carried out in like manner as described above with respect to PCR.

It will be readily appreciated that the detecting steps described herein may be carried out directly or indirectly. Thus, for example, if either wild-type PPI-1 or mutated PPI-1 is detected in the subject, then it is determined that the subject is not homozygous for either wild-type or mutated PPI-1. Kits for determining if a subject is or was afflicted with or is or was at increased risk of developing NIDDM will include at least one reagent specific for detecting for the presence or absence of mutated PPI-1 and instructions for observing that the subject is or was afflicted with or is or was at increased risk of developing NIDDM if the presence of a mutated PPI- 1 is detected. The kit may optionally include a nucleic acid for detection of the gene.

The present invention is explained in greater detail in the following non-limiting Examples.

Example 1 Materials Restriction enzymes, isopropyl 1-thio-β-D- galactopyranoside (IPTG), human thrombin and Staphylococcus aureus V8 protease (endoproteinase Glu-C) were purchased from Boehringer Mannheim. Glutathione- Sepharose were purchased from Pharmacia. Phosphorylase b and phosphorylase kinase were obtained from GIBCO-BRL. [γ- ³²P]ATP (>4,000 Ci/mmol) and [α-³²P]dCTP (>3,000 Ci/mmol) were purchased from ICN. Sequenase II was purchased from U.S. Biochemicals. Catalytic subunits of PP1 and PP2A, I- 1, and the catalytic subunit of cAMP-dependent protein kinase were purified from rabbit skeletal muscle. The polyclonal antibody against rabbit I-l (G185) was provided by Drs . P. Greengard and A.C. Nairn, Rockefeller University. Bovine anhydrotrypsin-Sepharose was provided by Drs. T. Kumazaki and H. Yokosawa of Hokkaido University, Sapporo, Japan.

Protein concentration was determined by the method of Bradford (Anal . Biochem . 72, 248-254 (1976)) using BSA as standard (E^1* ₂so =6 . 54 ) .

Example 2 Molecular Cloning of Human I-l cDNA

The human brain cDNA library constructed in λZAP

II (Stratagene) was provided by Dr. R. Joho, University of

Texas Southwestern Medical School. The cDNA library was transformed into E. coli BB4 grown in 1.0 % (w/v) Bacto- tryptone (Difco) , 0.5% (w/v) yeast extract (Difco) and

0.5% (w/v) NaCl at pH 7.5 at 42°C. Nitrocellulose filter lifts with denatured phage DNA were prehybridized in 6X SSC, 5X Denhardt ' s solution, 0.1% (w/v) sodium dodecyl sulphate (SDS), 0.3 M sodium phosphate pH 6.8 and 100 μg denatured salmon sperm DNA/ml. The filters were hybridized overnight at 42°C with ³²P-labelled 168 bp EcoRI-BamRl restriction fragment (0.5 x 10⁶ cpm/ml) from rat I-l cDNA random-primed with -³²p-dCTP. The filters were washed four times with 2X SSC and 0.1% (w/v) SDS at room temperature for 15 min each, followed by four washes with IX SSC and 0.1% (w/v) SDS at 50°C for 15 min each. Positive plaques were identified by aligning duplicate filters subjected to autoradiography . Individual clones were purified through secondary and tertiary screens, excised in vivo using helper phage, transferred to E. coli XLl-blue and characterized by double-stranded DNA sequencing using Sequenase II.

Example 3 Bacterial Expression of Human I-l

The NcoI-BstXI fragment of human I-l was excised from pBluescript SK-, blunt-ended with the Klenow fragment and subcloned into the Smal site in pGEX-2T (Pharmacia) . The plasmids were transformed into competent E. coli BL21 (Novagen) , JM109 (Promega) or DH5α (GIBCO-BRL) to express I-l as a fusion protein with glutathione-S-transferase ( GST ) .

A seed culture (5 ml) of E . coli was grown overnight at 37°C in Terrific Broth (see K. Tartof & C. Hobbs, BRL Focus 9(2), 12-14 (1987)) containing ampicillin (50 μg/ml) . The seed culture was added to 250 ml of medium maintained at 37 °C and bacterial growth was continued until an Aδoo of 0.6 was reached. The culture was cooled to 25°C and bacterial growth was allowed to proceed until Aβoo reached 0.8. Isopropyl 1-thio-β-D-galactopyranoside (IPTG) was then added to a final concentration of 1 mM and GST-I-

1 was induced by incubation at 25°C for 3 hr. The bacteria was centrifuged at 3,000 x g for 15 min and lysed by sonication in 20 ml of 50 mM Tris-HCl pH 7.5 containing 1%

(v/v) Nonidet P-40 (NP-40) , 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 1 mM PMSF at 4°C. The bacterial lysate was gently shaken with glutathione-Sepharose (10 ml) equilibrated in 20 mM Tris-HCl pH 7.5 containing 0.15 M NaCl (TBS) for 5 min at 4°C. The affinity matrix was washed with lysis buffer (2 x 20 ml) , followed by TBS containing 1% (v/v) NP-40 (3 x 30 ml) . GST-I-1 was eluted with 50 mM Tris-HCl pH 8.5 containing 10 mM glutathione and dialyzed against 0.5 mM Tris-HCl pH 7.5 containing 0.005% (w/v) Brij 35.

GST-I-1 (3-4 mg total protein) was applied to a 9% preparative SDS-PAGE (gel volume 60 ml) using Prep-Cell 490 (Bio-Rad) and subjected to electrophoresis at 50 mA constant current at room temperature for 17 hr. Fractions were analyzed by SDS-PAGE and GST-I-1 (apparent Mr 47 kDa) was pooled and dialyzed against 0.5 mM Tris-HCl pH 7.5, containing 0.005% (w/v) Brij 35 at 4°C. Purified GST-I-1 was lyophilized and stored at -80°C.

GST-I-1 (5 mg) was digested with thrombin (0.5- 1.0 NIH Unit/ml) in 10 ml of 50 mM Tris-HCl pH 8.5, containing 5 mM CaCl₂ at 30°C for 30 min. The reaction was terminated by adding PMSF (1 mM) followed by heating in a boiling water bath for 5 min. The reaction mixture was centrifuged at 40,000 x g for 20 min and the supernatant was subjected to preparative SDS-PAGE as described above. Fractions containing a single 28 kDa polypeptide representing human I-l were pooled, dialyzed and freeze- dried.

Example 4 Bacterial Expression of I-l Peptides

A synthetic gene (154 bp) was constructed using overlapping synthetic oligonucleotides that encoded 1-1(9- 54), as described by S. Shenolikar et al . (unpublished data). The EcoRI-BamRI fragment representing the 1-1(9-54) synthetic gene and a N-terminal cleavage site for Factor Xa was inserted into pRIT-2T (Pharmacia) vector. E. coli N4830 (Pharmacia) were transformed the expression plasmid and grown overnight in LB broth (250 ml) containing ampicillin (50 μg/ml) at 30°C. An equal volume of LB maintained at 54 °C was added and bacterial growth continued at 42°C for 2 hr. Bacteria were harvested by centrifugation at 3,000 x g for 15 min and lysed by sonication in 20 ml of 50 mM Tris-HCl pH 7.5 containing 1% (v/v) NP-40, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 1 mM PMSF at 4°C.

The bacterial lysate was shaken with 10 mis of IgG-Sepharose (Pharmacia) for 10 min at 4°C. The gel was washed with PBS (3 x 30 mis) and protein A-I-l(9-54) fusion protein was eluted with 0.2 M glycine-HCl pH 2.3(2 x 10 mis) . The fusion protein was dialyzed overnight against 50 mM Tris-HCl pH 7.5 containing 0.005% (w/v) Brij 35 and digested with Factor Xa (2U per mg of fusion protein) at 37°C for 90 min. The digest was heated in a boiling water bath for 5 min and denatured proteins removed by centrifugation at 30,000 x g for 30 min. The supernatant containing the 1-1(9-54) peptide was stored at

-20°C. Example 5 Site-directed Mutagenesis The NcoI-EcoRI insert from pGEX-2T was subcloned into pGEM-3Zf(-) (Promega) . Site directed mutagenesis was performed by PCR, with a "forward" primer and the SP6 primer in one reaction and a "backward" primer and the T7 primer in another reaction. 5 ' -GCCGCCCCGCCCCTGCC-3 ' (SEQ ID NO: 3) and 5 ' -GGCAGGGGCGGGGCGGC-3 ' (SEQ ID NO: 4) were used as "forward" and "backward" primers to substitute alanine in place of the phosphoacceptor, threonine-35. 5'-GCCGCCCCGACCCTGCC-3' (SEQ ID NO:5) and 5'- GGCAGGGTCGGGGCGGC-3' (SEQ ID NO: 6) substituted aspartic acid in this position. PCR reactions (35 cycles) were undertaken at 94°C for 30 sec, 1 min at 55°C and 1 min at

72°C. PCR products were purified by electrophoresis in 1%

(w/v) agarose. Equimolar amounts of the two products were mixed and further amplified using only external T7 and SP6 primers. The second PCR product was digested with Ncol and EcoRI and subcloned into pGEM-3Zf(-). Mutations were verified by double-stranded sequencing using Sequenase II.

Example 6 Phosphorylation of I-l by cAMP-dependent Protein Kinase

I-l (0.25 mg/ml) or GST-I-1 (0.5 mg/ml) was phosphorylated with purified catalytic subunit of cAMP- dependent protein kinase (0.2 μg/ml) in 50 mM Tris-HCl pH

7.5 containing 100 μM ATP and 1 mM MgCl₂ at 30°C for 1 to 4 hr. The extent of I-l phosphorylation was monitored by including trace amounts of [γ-³²P]ATP in the reaction. Incorporation of ³²P-phosphate into I-l was followed by SDS-PAGE and autoradiography or by trichloroacetic acid (15%, w/v) precipitation. Concentration of I-l was established by the incorporation of 1 mole of ³²P-phosphate into 1 mole of protein. Example 7 Protein Phosphatase Assay ³²P-phosphorylase a was phosphorylated with phosphorylase kinase as described in S. Shenolikar and T. Ingebritsen, Methods Enzymol . 107, 102-129 (1984). Concentration of phosphorylase was determined using E^1% ₂so of 13.1. PP1 and PP2A were incubated with 10 μM ³²P- phosphorylase a in 50 mM Tris-HCl pH 7.0, 1 mg/ml BSA, 1 mM EDTA, 0.3 % (v/v) 2-mercaptoethanol (total volume 60 μl) at 30°C. Reaction was terminated by adding 0.1 ml of 20% (w/v) trichloroacetic acid and 0.1 ml of BSA (6 mg/ml), cooled on ice for 5 min and centrifuged at 15,000 x g for 5 min. ³²P-phosphate release was monitored by mixing 200 μl of supernatant with 2 mis of Safety-Solve scintillation fluid (RPI) and counting in a liquid scintillation counter. One unit of phosphatase was defined as the release of 1 nmole of phosphate in 1 min under the assay conditions . One unit of I-l activity was defined as the amount required to inhibit 0.02 U of PP1 by 50 % in this assay.

Example 8 Purification of I-l Peptides

I-l peptides were purified by preparative SDS- PAGE followed by reversed-phase HPLC on Ci8~μbondapak (300 x 7.5 mm, Waters) eluted with 0.1% (v/v) trifluoroacetic acid in water using a linear gradient of 1 to 65% (v/v) acetonitrile . Fractions were pooled, dried and redissolved in 1.0 mM Tris-HCl pH 7.5 containing 0.005% (w/v) Brij 35.

For mass spectrometry, I-l peptides were further separated by reversed-phase HPLC using Cs (5 μm) -Deltabond- LC column (100 x 1 mm, Keystone Scientific) equilibrated with 0.08% (v/v) trifluoroacetic acid in ultrapure water (Solvent A) . Peptides were eluted with a linear gradient of 10 to 70% solvent B (90% (v/v) acetonitrile in solvent A) at a flow rate of 50 μl/min. The purified peptides were vacuum-dried and resuspended in 50% aqueous acetonitrile containing 1% (v/v) formic acid for electrospray mass spectrometry.

Example 9 Sequence Analysis of I-l Protein and Peptides I-l proteins and peptides were separated by SDS-

PAGE and electrophoretically transferred to Trans-Blot membrane (Bio-Rad) . The membranes were stained with Coomassie Blue and protein bands were excised for N- terminal sequence determination using the Applied Biosystems Model 477A peptide sequencer.

C-terminal sequences were determined by digestion of selected I-l peptides with Staphylococcus V8 protease (1%, w/w) in 50 mM ammonium bicarbonate pH 8.6 at 37°C. The digest was adjusted to pH 5.0 using 1 mM HC1 to terminate the reaction. An equal volume of 50 mM sodium acetate pH 5.0 containing 20 mM CaCl₂ was added and the sample was applied to anhydrotrypsin-Sepharose . The column was washed with 50 mM sodium acetate pH 5.0 containing 20 mM CaCl₂ and bound peptides eluted with 5 mM HC1. See, H. Yokosawa & S. Ishii, Biochem . Biophys . Res . Commun . 72, 1443-1449 (1976); S. Ishii et al., Methods Enzymol . 91, 378-383 (1983) . The peptides were lyophilized, redissolved in 0.1 % trifluoroacetic acid and subjected to reversed- phase HPLC on a Vydac Cis column prior to sequence determination.

Example 10 Electrospray Ionization Mass Spectrometry I-l proteins and peptides were analyzed using

Fisons-VG Quattro BQ triple quadrupole mass spectrometer equipped with a pneumatically assisted electrostatic ion source operating at atmospheric pressure. Samples were introduced by loop injection into a stream of 50% aqueous acetonitrile containing 1% (v/v) formic acid at 6 μl/min and spectra acquired in the multichannel analyzer mode from m/e 700-1400 with a scan time of 10 sec. Mass spectra were transformed to a molecular mass scale calibrated with equine cardiac myoglobin (Mr 16,951.48) with resolution corresponding to peak width at half height of 1.4 Da for m/e 893.

Example 11

Biosensor Analysis

Phosphatase/inhibitor interactions were analyzed using Fisons IAsys optical biosensor system. Affinity- purified polyclonal antibody (16 μg protein) against

Schistosomal glutathione-S-transferase (GST) was covalently linked to an IAsys cuvette activated with EDC (l-Ethyl-3- [3-dimethylaminopropyl] -carbodiimide hydrochloride) - NHS (N-hydroxysuccinimide) in 10 mM acetate buffer pH 4.5 (Fisons application note 3.1). Residual reactive groups were blocked with 1 M ethanolamine pH 8.5 and unbound protein removed by washing with 10 mM HC1. The cuvette was equilibrated with phosphate-buffered saline (10 mM phosphate, 2.7 mM KCl and 137 mM NaCl, pH 7.4) containing 0.05% (v/v) Tween 20 (PBS-

Tween) prior to loading GST-I-1 (10 μg) . Time-dependent binding of GST-I-1 to the immobilized anti-GST antibody was monitored as an increase in the evanescent response

(measured in arc seconds) . Excess GST-I-1 was removed with PBS-Tween. Purified PP1 (or PP2A) catalytic subunits were added, and binding was observed as a time-dependent increase in the evanescent field response. The cuvette was then washed with PBS-Tween and dissociation of the phosphatase from GST-I-1 was analyzed. The IAsys cuvette was regenerated with 10 mM HC1 prior to binding phosphorylated GST-I-1 to the immobilized anti-GST antibody. PP1 (or PP2A) binding to phosphorylated GST-I-1 was analyzed as described .

Example 12 Circular Dichroism Analyses

Purified I-l proteins and peptides (1 mg protein/ml of PBS) were analyzed for their secondary structure using an Aviv circular dichroism spectrophotometer model 62DS (Lakewood, N.J.). Phosphorylated and unphosphorylated I-l proteins and peptides were scanned at wavelength 180 to 320 n with a path length of 1 cm and slit width between 0.25 to 0.3 mm. CD spectra at a range of temperatures between 10°C and 50°C were undertaken with the I-l proteins and peptides. to examine the stability of their secondary structure.

Example 13 Western Immunoblot Analysis SDS-PAGE was carried out by the method of

Laemmli, Na ture 227, 680-685 (1970) . Proteins were transferred to a Trans-Blot membrane (Bio-Rad) at 100 V for 1 hr in 25 mM Tris/192 mM glycine pH 8.3 containing 20% (v/v) methanol. The membranes were blocked with 1% (w/v) dried milk in 20 mM Tris-HCl pH 7.5 containing 0.1% (v/v) Tween 20 and 150 mM NaCl (TTBS) for 1 hr at room temperature prior to incubation with a polyclonal antibody generated against rabbit I-l, diluted 1/500 or 1/1000 in TTBS, for 1 hr at room temperature or overnight at 4°C. Immunoblots were washed with TTBS and incubated with anti- rabbit IgG linked to horseradish peroxidase in TTBS for 1 hr at room temperature. Immunodetection of I-l was carried out using the ECL system (Amersham) or the color reaction developed with 4-chloro-l-naphtol and H₂0₂. Example 14 Molecular Cloning of Human Brain I-l cDNA

A total of 900,000 recombinants were screened from a human brain cDNA library and 14 positive clones were obtained. Eight of these were further purified through secondary and tertiary screens and all contained an EcoRI insert of approximately 700 bp. Three independent clones were sequenced in both directions and yielded the identical nucleotide sequence that terminated with the 3'- poly (A) ⁺ tail. The cDNA predicted a polypeptide of 171 amino acids with 85% overall sequence identity to rabbit

(Aitken et al., supra ) and rat I-l (Elbrecht et al, supra ) . C-terminal sequences of the three I-l proteins showed considerable differences (37% identity in the last 51 residues) . Indeed, rat and human I-l contained a five amino acid insert near the C-terminus not found in rabbit I-l. This indicates the remarkable conservation of the N- terminal region (97% identity in the first 58 residues) containing the threonine phosphorylated by PKA (see P. Cohen et al., FEBS Lett . 76, 182-186 (1977)).

EXAMPLE 15 Characterization of Recombinant Human I-l

Maximum expression of GST-I-1 (apparent M_r 47 kDa on SDS-PAGE) in several strains of E. coli was obtained by 2-3 hr growth in media containing IPTG. The fusion protein was rapidly purified from bacterial extracts by affinity chromatography on glutathione-

Sepharose (Figure 1A, lane 2) . This was followed by preparative SDS-PAGE which purified GST-I-1 to homogeneity

(Figure 1A, lane 3) . The fusion protein was phosphorylated by PKA (Figure IB) to a stoichiometry of 1 mole of phosphate per mole of protein and phosphoamino acid analysis established that the modification occurred exclusively on threonine (data not shown) . For most studies, GST-I-1 in the E. coli BL21 strain that showed significantly reduced degradation of the fusion protein were expressed. A typical 250 ml culture of BL21 bacteria yielded 7-9 mg of GST-I-1.

GST-I-1 was digested with thrombin and I-l was purified by preparative SDS-PAGE. Human I-l migrated on SDS-PAGE with an apparent molecular weight of 28 kDa slightly larger than the rabbit skeletal muscle I-l (Figure 2A) and readily cross-reacted with a polyclonal antibody generated against the rabbit I-l (Figure 2B) . Mass spectrometry of the recombinant human I-l confirmed its molecular size at 19,179 precisely as predicted by the cDNA. Thus, the anomalous electrophoretic mobility of rabbit, rat and human I-l on SDS-PAGE most likely reflects low detergent binding [Nimmo and Cohen, supra 1978a] . Following stoichiometric phosphorylation (1 mole of phosphate /mole of protein) by PKA, GST-I-1 inhibited PPl activity with an IC₅₀ of approximately 30 nM (see Table 1, below). More than 1, 000-fold higher concentration of phosphorylated GST-I-1 was required to inhibit PP2A. Thrombin cleavage yielded free human I-l and increased its potency as a PPl inhibitor to that of I-l purified from rabbit skeletal muscle (IC50 1 nM) . Phosphorylated rabbit and human I-l also inhibited PP2A (the major type-2 phosphatase in mammalian tissues) , albeit at concentrations exceeding 20 μM. Neither PPl nor PP2A was inhibited by dephosphorylated rabbit and human I-l (data not shown) . Thus, the overall functional properties of recombinant human I-l were similar to I-l purified from tissues . TABLE 1 . Inhibition of T e-1 and T pe-2 Phosphatases¹

babbit skeletal muscle 1-1 was purified according to Cohen et al [1988 supra]. Human 1-1 fused to GST was expressed in E.coli The fusion protein was digested with thrombin and the 1-1 proteins and peptides purified to homogeneity as described in Methods 1-1 was phosphorylated with PKA prior to analyzing the inhibition of purified PP1 and PP2A catalytic subunits using ³²P-phosphorylase a as substrate. IC₅₀ values were calculated from an average of 5 independent experiments. 1-1 peptide a (-3-61 ) and peptide b (9-61 ) were analyzed in 3 and 2 separate experiments respectively. All values are presented with standard errors

When pGEX-2T-hI-l was transformed in E. coli JM109 or DH5 and the bacteria were grown at 37°C in the presence of IPTG, a significantly smaller fusion protein was expressed. Chromatography of JM109 or DH5α extracts on glutathione-agarose yielded a polypeptide of an apparent Mr 37 kDa by SDS-PAGE. Following thrombin digestion, two peptides of apparent Mr 14 and 12 kDa were obtained (Figure 3A) . Both were readily phosphorylated by PKA and the mixture of phosphopeptides inhibited PPl. Thus, the individual I-l peptides (a and b) were purified by preparative SDS-PAGE and reversed-phase HPLC. Peptide a yielded the N-terminal sequence, GSPMEQDNSRKIQF (SEQ ID NO: 7) and corresponded to N-terminus of human I-l with the additional three amino acids derived from the pGEX-2T linker (Figure 3C) . Peptide b had the sequence, KIQFTVPLLEPHLDP (SEQ ID NO: 8) and represented a cleavage between residues 8 and 9 in human I-l. Molecular mass for peptides a and b was estimated by mass spectrometry as 7,265.82 and 6,066.38 respectively (Figure 3C) . This suggested that both peptides terminated at lysine-61, which was confirmed by digesting peptides a and b with Staphylococcus V8 protease and chromatography on anhydrotrypsin-Sepharose. The predicted C-terminal lysine-containing peptides were eluted from the affinity matrix and further purified by HPLC. In both cases, a major peptide (Figure 3B) with the sequence, DRIPNPHLK (SEQ ID NO:9), was obtained. This established that peptides a and b terminated at lysine-61 and molecular weights calculated from their amino acid sequence precisely matched those determined by mass spectrometry (Figure 3C) •

Following PKA phosphorylation, peptide a (I-l(- 3-61)) inhibited PPl with IC₅₀ of 3.7 nM and, peptide b (I- 1(9-61)) inhibited with an IC₅o of 4.5 nM. Thus, the two N-terminal peptides of human I-l were nearly as potent as full-length I-l in inhibiting PPl activity. Neither phosphopeptide inhibited PP2A activity at the highest concentration examined (10 μM) .

EXAMPLE 16

Mutagenesis of the Phosphoacceptor Site at Threonine- 35

Site-directed mutagenesis was used to substitute a non-phosphorylated residue, alanine, in place of the phosphoacceptor at threonine-35 and establish the role of

PKA phosphorylation for I-l function. GST-I-1 (T35A) expressed in E. coli BL21 was purified to homogeneity

(Figure 3A) , using immunoblotting with the polyclonal antibody against rabbit I-l to monitor the purification.

As anticipated, GST-I-1 (T35A) was not phosphorylated by

PKA and at 50 μM, the highest concentration of the fusion protein that did not aggregate during the assay, GST-I-

1(T35A) had no effect on PPl or PP2A activity (see Table 2, below) . Thrombin digestion of the fusion protein and subsequent purification of I-l (T35A) further confirmed that the mutant protein was inactive as a phosphatase inhibitor. These data established that PKA phosphorylation of threonine-35 was essential for I-l to inhibit PPl at nanomolar concentrations and PP2A at micromolar concentrations .

TABLE 2. Mutations of the Phosphoacceptor Site in Human Inhibitor-1

'Site-directed mutagenesis was used to substitute alanine or aspartic acid in place of threonιne-35. Mutant GST-I- 1 (T35A) and GST-I-1 (T35D) were expressed in E. coli and purified as described in Methods. 1-1 proteins and peptides were analyzed for inhibition of PP1 and PP2A catalytic subunits. Each result represents an average of 3 independent experiments and is shown with standard errors. *The HPLC-punfied 1-1 (2-65, T35D) was assayed in triplicate.

To mimic the functional effects of phosphorylation, an aspartic acid was substituted in place of the phosphoacceptor. In contrast to GST-I-1 (T35A) that at 50 μM did not inhibit PPl or PP2A, GST-I-1 (T35D) was more effective and a constitutive inhibitor of these phosphatases (Table 2) . However, the efficacy of GST-I- 1(T35D) as a PPl inhibitor (IC50 30μM) was very similar to PP2A (IC50 44 μM) . Following thrombin cleavage, the purified I-l (T35D) no longer distinguished between PPl and PP2A (IC50 24-25 μM) . Earlier studies [S. Shenolikar et al., Biochem . Soc . Trans . 6:935-937 (1978)] used CNBr cleavage to isolate an active phosphopeptide of rabbit I- 1. CNBr cleavage of GST-I-1 (T35D) increased its potency as a PPl inhibitor. HPLC purified 1-1(2-65, T35D) inhibited PPl with IC50 235 nM, an 100-fold increase over the activity of the parent I-l (T35D) protein. Moreover, the I- 1(2-65, T35D) peptide at 10 μM completely inhibited PPl but only resulted in a 10% decrease in PP2A activity. These data provided new insight into the conformation of intact I-l induced by phosphorylation that yields a potent and selective PPl inhibitor. Moreover, functions associated with this phosphorylation-induced conformation were not changed by truncation to the N-terminal functional domain. By comparison, substitution of an acidic residue in place of the phosphoacceptor had different functional effects in the context of the full- length I-l and the N-terminal peptide.

Example 17 Circular Dichroism Analysis of Recombinant I-l Protein and Peptides .

Earlier studies { see, e . g. , P. Cohen et al.,

FEBS Symp. 54: 161-169 (1979)) used circular dichroism to show that I-l isolated from boiled skeletal muscle extracts was largely disordered in structure. PKA phosphorylation activated the rabbit I-l as a PPl inhibitor but no effect on its limited secondary structure that could be discerned by denaturation with 6M guanidine hydrochloride. Circular dichroism showed that recombinant human I-l was also largely disordered in solution. Stoichiometric phosphorylation did not result in a significant change in its CD spectrum (data not shown) . Comparison of spectra at temperatures from 10 to 50°C showed that the higher temperatures increased in the extent of random coil and established that both phosphorylated and unphosphorylated I-l contained limited secondary structure. However, no difference in structure could be attributed to covalent modification (data not shown). Surprisingly, I-l (-3-61) that eliminated 110 residues from the C-terminus of I-l protein was also largely disordered. Moreover, like full length I-l, the overall secondary structure of the functional domain represented in I-l (-3-61) was unchanged by phosphorylation . Example 18 Association of I-l with PPl Catalytic Subunit The Fisons IAsys optical biosensor system was used to investigate the binding of phosphorylated and unphosphorylated I-l to purified PPl and PP2A catalytic subunits. A polyclonal anti-GST antibody was covalently coupled to the biosensor surface and used to subsequently immobilize either unphosphorylated or phosphorylated GST- I-1. Excess fusion protein was removed with PBS-Tween. Time-dependent increase in the evanescent field response following the binding of purified PPl catalytic subunit (10 μg protein) to the immobilized GST-I-1 saturated at a value 8-10-fold higher than the maximum response evoked by a similar amount of PP2A catalytic subunit. This suggested that compared to PPl, less than 10% of PP2A bound to the immobilized GST-I-1. Neither PPl nor PP2A bound to the biosensor surface when GST alone was immobilized, indicating that the association of the phosphatases was mediated through I-l. PPl dissociated slowly from GST-I-1. While phosphorylation of GST-I-1 increased its phosphatase inhibitor activity by 1, 000- fold, it slowed the dissociation of the PPl catalytic subunit by only 2-fold. The K₀ff values for the PP2A catalytic subunits from either phosphorylated or unphosphorylated GST-I-1 were approximately 90-fold faster than those for PPl, as shown below in Table 3. The slow off-rate of the PPl catalytic subunit may be an important determinant of I-l's specificity as a phosphatase inhibitor. The dissociation constants were calculated using an average of 260 data points and were corrected for the very slow dissociation of GST-I-1 from the anti-GST antibody. Table 3. Kof values for the PP2A catalytic subunits from either phosphorylated or unphosphorylated GST-I-1

However, the biosensor studies showed that PPl binding was largely independent of I-l's phosphorylation state and by itself may not account for inhibition of this phosphatase.

Example 19

An N-terminal Truncation Inactivates I-l

Earlier studies used proteolysis of rabbit I-l to isolate the "minimal" active fragment, 1-1(9-54). This sequence was found to be entirely conserved in human I-l and also shared homology with DARPP-32, a neuronal cAMP- regulated phosphoprotein that also inhibited PPl (Figure 4). 1-1(9-54) was expressed in E. coli N4830 as a fusion protein with the IgG-binding domain of Staphylococcus aureus protein A, as described in Example 1. The fusion protein was purified on IgG-Sepharose but was not phosphorylated in vi tro by PKA. Thus, the ability of the fusion protein to inhibit PPl activity could not be analyzed. Digestion with factor Xa at a unique site constructed in the linker yielded the 1-1(9-54) peptide with two additional N-terminal residues. This peptide was readily phosphorylated by PKA and following phosphorylation, inhibited PPl activity in a dose- dependent manner (Figure 4) . As expected, the unphosphorylated peptide was an ineffective PPl inhibitor. EcoRl-Kpnl cleavage deleted the nucleotide sequence encoding the tetrapeptide, KIQF, conserved in I-l and DARPP-32. Following cleavage of the altered fusion protein with Factor Xa, 1-1(13-54) with four additional N-terminal residues was obtained. 1-1(13-54) was phosphorylated by PKA with similar efficiency to 1-1(9-54) but the resulting phosphopeptide was a very poor inhibitor of PPl activity (Figure 4) . This suggested that the KIQF sequence was required in addition to phosphorylation of threonine-35 for PPl inhibition.

Protein A-I-l(9-54) was immobilized to the biosensor surface via the anti-GST antibody. However, no significant binding of PPl or PP2A catalytic subunits was observed to this fusion protein. Hence, the fusion of I- 1(9-54), to protein A not only impaired its phosphorylation by PKA but also prevented PPl binding.

The foregoing Examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Shenolikar, Shirish

Means, Anthony

(ii) TITLE OF INVENTION: Human Phosphatase Inhibitor-1 Gene and Methods of Screening for

Non-Insulin Dependent Diabetes Mellitus

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sorojini J. Biswas

(B) STREET: Post Office Box 37428

(C) CITY: Raleigh

(D) STATE: North Carolina

(E) COUNTRY: USA

(F) ZIP: 27627

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: BISWAS, Sorojini J.

(B) REGISTRATION NUMBER: 39,111

(C) REFERENCE/DOCKET NUMBER: 5405-121 WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (919) 854-1400

(B) TELEFAX: (919) 854-1401

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 662 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..524

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

CCCCAGCC ATG GAG CAA GAC AAC AGC CCC CGA AAG ATC CAG TTC ACG GTC 50 Met Glu Gin Asp Asn Ser Pro Arg Lys He Gin Phe Thr Val 1 5 10

CCG CTG CTG GAG CCG CAC CTT GAC CCC GAG GCG GCG GAG CAG ATT CGG 98 Pro Leu Leu Glu Pro His Leu Asp Pro Glu Ala Ala Glu Gin He Arg 15 20 25 30

AGG CGC CGC CCC ACC CCT GCC ACC CTC GTG CTG ACC AGT GAC CAG TCA 146 Arg Arg Arg Pro Thr Pro Ala Thr Leu Val Leu Thr Ser Asp Gin Ser 35 40 45

TCC CCA GAG ATA GAT GAA GAC CGG ATC CCC AAC CCA CAT CTC AAG TCC 194 Ser Pro Glu He Asp Glu Asp Arg He Pro Asn Pro His Leu Lys Ser 50 55 60

ACT TTG GCA ATG TCT CCA CGG CAA CGG AAG AAG ATG ACA AGG ATC ACA 242 Thr Leu Ala Met Ser Pro Arg Gin Arg Lys Lys Met Thr Arg He Thr 65 70 75

CCC ACA ATG AAA GAG CTC CAG ATG ATG GTT GAA CAT CAC CTG GGG CAA 290 Pro Thr Met Lys Glu Leu Gin Met Met Val Glu His His Leu Gly Gin 80 85 90

CAG CAG CAA GGA GAG GAA CCT GAG GGG GCC GCT GAG AGC ACA GGA ACC 338 Gin Gin Gin Gly Glu Glu Pro Glu Gly Ala Ala Glu Ser Thr Gly Thr 95 100 105 110

CAG GAG TCC CGC CCA CCT GGG ATC CCA GAC ACA GAA GTG GAG TCA AGG 386 Gin Glu Ser Arg Pro Pro Gly He Pro Asp Thr Glu Val Glu Ser Arg 115 120 125 CTG GGC ACC TCT GGG ACA GCA AAA AAA ACT GCA GAA TGC ATC CCT AAA 434 Leu Gly Thr Ser Gly Thr Ala Lys Lys Thr Ala Glu Cys He Pro Lys 130 135 140

ACT CAC GAG AGA GGC AGT AAG GAA CCC AGC ACA AAA GAA CCC TCA ACC 482 Thr His Glu Arg Gly Ser Lys Glu Pro Ser Thr Lys Glu Pro Ser Thr 145 150 155

CAT ATA CCA CCA CTG GAT TCC AAG GGA GCC AAC TCG GTC TGA 524

His He Pro Pro Leu Asp Ser Lys Gly Ala Asn Ser Val * 160 165 170

GAGAGGAGGA GGTATCTTGG GATCAAGACT GCAGTTTGGG AATGCATGGA CACCGGATTT 584

GTTTCTTAπ CCTTCACTTT TGGGGAAAAT CTCTTGTTTT TAAAAAGTGA TAAATTTGGT 644

GTTAGGTCAA AAAAAAAA 662

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 172 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Gin Asp Asn Ser Pro Arg Lys He Gin Phe Thr Val Pro Leu 1 5 10 15

Leu Glu Pro His Leu Asp Pro Glu Ala Ala Glu Gin He Arg Arg Arg 20 25 30

Arg Pro Thr Pro Ala Thr Leu Val Leu Thr Ser Asp Gin Ser Ser Pro 35 40 45

Glu He Asp Glu Asp Arg He Pro Asn Pro His Leu Lys Ser Thr Leu 50 55 60

Ala Met Ser Pro Arg Gin Arg Lys Lys Met Thr Arg He Thr Pro Thr 65 70 75 80 Met Lys Glu Leu Gin Met Met Val Glu His His Leu Gly Gin Gin Gin 85 90 95

Gin Gly Glu Glu Pro Glu Gly Ala Ala Glu Ser Thr Gly Thr Gin Glu 100 105 110

Ser Arg Pro Pro Gly He Pro Asp Thr Glu Val Glu Ser Arg Leu Gly 115 120 125

Thr Ser Gly Thr Ala Lys Lys Thr Ala Glu Cys He Pro Lys Thr His 130 135 140

Glu Arg Gly Ser Lys Glu Pro Ser Thr Lys Glu Pro Ser Thr His He 145 150 155 160

Pro Pro Leu Asp Ser Lys Gly Ala Asn Ser Val * 165 170

Claims

That Which Is Claimed Is:

1. Isolated DNA encoding human protein phosphatase inhibitor-1.

2. Isolated DNA according to claim 1 encoding human protein phosphatase inhibitor-1 having the amino acid sequence of SEQ ID NO: 2 .

3. Isolated DNA according to claim 1 having the nucleotide sequence according to SEQ ID NO: 1.

4. A vector comprising DNA according to claim

5. A host cell containing a vector according to claim 4.

6. An oligonucleotide probe that specifically binds to a DNA according to claim 1.

7. A method of detecting DNA encoding human protein phosphatase inhibitor-1 (PPI-1) in a sample DNA, comprising: contacting an oligonucleotide probe that specifically binds to DNA encoding PPI-1 to said sample DNA, and then detecting the presence or absence of binding of said oligonucleotide probe to said sample DNA, the presence of binding indicating the presence of DNA encoding human PPI-1 in said sample.

8. A method for screening a subject for increased risk of non-insulin dependent diabetes mellitus (NIDDM) , comprising: detecting the presence or absence of an inactivating mutation in the protein phosphatase inhibitor-1 (PPI-1) gene in said subject; and observing whether or not the subject is at increased risk of NIDDM by observing if a PPI-1 mutation is or is not detected, wherein the presence of a PPI-1 mutation indicates said subject is at increased risk for NIDDM.

9. A method according to claim 8, wherein said detecting step is carried out by collecting a biological sample containing DNA from said subject, and then determining the presence or absence of said mutation in said biological sample.

10. A method according to claim 9, wherein said determining step is carried out by amplifying DNA encoding PPI-1.

11. A method according to claim 10, wherein said amplifying step is carried out by polymerase chain reaction.

12. A method according to claim 10, wherein said amplifying step is carried out by ligase chain reaction.

13. A method according to claim 8, wherein said detecting step comprises detecting whether said subject is homozygous for the gene encoding PPI-1.

14. A method according to claim 8, wherein said subject is a human subject.

15. A method according to claim 8, wherein said mutation is a substitution mutation.

16. A method according to Claim 8, wherein said mutation is a mutation of the phosphoacceptor site at the threonine-35 position of PPI-1.

17. A method according to claim 8, wherein said mutation is the substitution of a non-phosphorylated residue for threonine at the threonine-35 position of PPI- 1.

18. A method according to claim 16 wherein said non-phosphorylated residue is alanine.

19. A method according to claim 8, wherein said mutation is a frame-shift mutation at codon 10 of the gene encoding PPI-1.

20. A method according to claim 8, wherein said mutation is a mutation at base 435 of the gene encoding

PPI-1.

21. A method according to claim 20, wherein said mutation is a substitution mutation, and wherein the nucleotide at base 435 is substituted with a ^XG' nucleotide.