WO2000071080A9 - Interaction of the cyclic-amp-specific phosphodiesterase pde4d5 with rack1 - Google Patents
Interaction of the cyclic-amp-specific phosphodiesterase pde4d5 with rack1Info
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- WO2000071080A9 WO2000071080A9 PCT/US2000/013961 US0013961W WO0071080A9 WO 2000071080 A9 WO2000071080 A9 WO 2000071080A9 US 0013961 W US0013961 W US 0013961W WO 0071080 A9 WO0071080 A9 WO 0071080A9
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- rackl
- pde4d5
- seq
- peptide
- interaction
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
Definitions
- This invention relates to drug development. More particularly, the invention relates to type-specific modulators of cyclic AMP-specific phosphodiesterases . Modulation of the levels of the second messenger adenosine cyclic 3', 5'-monophosphate (cyclic AMP or cAMP) in cells is important in the regulation of numerous physiological processes, including those in the immune/inflammatory systems, vascular smooth muscle, and the brain.
- cyclic AMP or cAMP second messenger adenosine cyclic 3', 5'-monophosphate
- Cyclic nucleotide phosphodiesterases are a diverse family of enzymes that hydrolyze cAMP and guanosine cyclic 3', 5'-monophosphate (cyclic GMP or cGMP) and thus play an important role in modulating cAMP levels. J.A. Beavo, 75 Physiol. Rev. 725-748 (1995) .
- the cAMP-specific phosphodiesterases (PDE4s) can be differentiated from other PDEs by sequence homology of the catalytic region of the enzymes, M.D. Houslay et al . , 44 Advances in Pharmacology 225-342 (1998) , and by their ability to be specifically inhibited by the drug 4- [3- (cyclopentyloxy) -4-methoxyphenyl] -2-pyrrolidinone
- Rolipram and other specific PDE4 inhibitors have been shown to have anti-depressant, anti-inflammatory, and smooth-muscle relaxant activity in humans. M.D. Houslay et al., 44 Advances in Pharmacology 225-342 (1998).
- the PDE4 enzymes are also characterized by the presence of unique regions of amino acid sequence outside the catalytic region of the proteins, which are called upstream conserved regions 1 and 2 (UCR1 and UCR2) and are located in the amino-terminal half of the proteins.
- UCR1 and UCR2 upstream conserved regions 1 and 2
- the PDE4s are comprised of a large family of isoforms, encoded by four different genes ( PDE4A, PDE4B, PDE4C, and PDE4D) in humans, with additional diversity being generated by alternative mRNA splicing. M.D. Houslay et al., 44 Advances in Pharmacology 225-342 (1998) . All the catalytic regions of different isoforms that are encoded by the same gene are identical. In addition, the catalytic regions encoded by each of the four PDE4 genes are extremely similar. Therefore, drugs that act at the catalytic site of one of the PDE4 isoforms will inhibit all PDE4 isoforms. To develop isoform-specific inhibitors, compounds are identified that interact with unique region of each isoform, not with the catalytic region.
- the five isoforms differ by the substitution of unique blocks of amino acids at the amino-terminal regions of their respective proteins.
- the two smaller PDE4D isoforms, PDE4D1 and PDE4D2 are located exclusively in the cytosolic fraction of the cell.
- the larger isoforms PDE4D3, PDE4D4, and PDE4D5 are each found both in the cytosol as well as in association with cellular particulate fractions.
- the functional consequences of this diversity are poorly understood.
- the PDE4D3 isoform is a substrate for protein kinase A (PKA) , which serves to activate this isoform.
- PPA protein kinase A
- the PDE inhibitor has been on the market for many years for the treatment of asthma and other pulmonary diseases.
- Theophylline inhibits members of all PDE families, and thus has a number of potential serious side effects.
- Recent drug development has focused on the development of type-specific inhibitors.
- the best-known type-specific inhibitor is the PDE5 inhibitor, sildenafil (VIAGRA, Pfizer Inc.), the well-known drug that is marketed for erectile dysfunction.
- Selective PDE4 inhibitors are being synthesized and evaluated by a large number of drug companies.
- PDE4 inhibitors such as the drug rolipram, have smooth-muscle relaxant, anti-inflammatory, pro-apoptotic, and immunomodulatory properties, and are being tested as therapeutic agents for a variety of diseases, including asthma, rheumatoid arthritis, and other disorders.
- the PDE4 inhibitor, ARIFLOW (SmithKline Beecham) is currently in Phase III trials for asthma. All PDE4 inhibitors that have been tested to date, however, inhibit all the various PDE4 isoforms relatively equally. This is because all the PDE4 inhibitors that have been tested to date work as competitive (or partially competitive) inhibitors of the catalytic site of the enzyme. Therefore, it would be advantageous to develop inhibitors that work outside of the catalytic site.
- compositions and methods for modulating the activity of cAMP-specific phosphodiesterases and methods for screening type-specific inhibitors of cAMP-specific phosphodiesterases would be a significant advancement in the art.
- compositions that modulate the activity of cAMP-specific phosphodiesterases .
- compositions that are type-specific modulators of cAMP- specific phosphodiesterases.
- the peptide is a member selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 8, and mixtures thereof.
- a method for altering the activity of a PDE4D5 that interacts with RACKl comprises contacting RACKl with a peptide that has a sequence that permits formation of a complex between the peptide and RACKl, wherein the complex mimics the interaction of PDE4D5 with RACKl, such that the complex is formed and formation of the complex inhibits the interaction between PDE4D5 and RACKl or stimulates the activity of PDE4D5.
- a peptide for inhibiting interaction between PDE4D5 and RACKl wherein the peptide is a member selected from the group consisting of SEQ ID NO: 18, SEQ ID NO:34, SEQ ID NO:48, and mixtures thereof.
- composition for pharmaceutical use of the peptide comprises an admixture of: (a) a peptide for inhibiting interaction between PDE4D5 and RACKl, wherein the peptide is a member selected from the group consisting of SEQ ID NO:18, SEQ ID NO:34, SEQ ID NO:48, and mixtures thereof; and (b) a pharmaceutically acceptable carrier.
- a method for treating a condition that is susceptible of being ameliorated by a type-specific inhibitor of PDE4D5 comprises administering an effective amount of a composition comprising:
- a peptide for inhibiting interaction between PDE4D5 and RACKl wherein the peptide is a member selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 48, and mixtures thereof; and (b) a pharmaceutically acceptable carrier.
- Yet another illustrative embodiment of the invention comprises a method for obtaining a peptide that inhibits interaction of PDE4D5 and RACKl comprising:
- FIG. 1 shows a schematic diagram of the amino-terminal region of PDE4D5.
- the line diagram at the top represents the five human PDE4D isoforms.
- the numbers 1-5 represent isoforms PDE4D1 through PDE4D5, respectively.
- the heavy bar indicates sequences homologous to those in other PDE4 isoforms, with the strongest regions of conservation (the catalytic region and
- the thin, branched lines adjacent to the numbers indicate sequence regions unique to each isoform. The thin lines merge where the sequences of the various isoforms join that of the shared sequence.
- the PDE4D2 isoform begins in the middle of UCR2, at a methionine that is internal to the other four isoforms.
- the bottom of the figure shows the amino acid sequence (in single-letter code) of the unique 88 ammo-terminal region of PDE4D5 (SEQ ID NO:l).
- the numbers immediately above the sequence indicate the ammo acid co-ordinates. Ammo acid residues subjected to site-directed mutagenesis (FIGS.
- FIGS. 6C and 7B, below are underlined (SEQ ID NO:2 through SEQ ID NO:ll).
- the small arrows immediately above the sequence indicate ammo acid residues that, when mutated, block the interaction with RACKl (FIGS. 6C and 7B, below) .
- the numbers below the sequence indicate the a mo- terminal ends of the nested deletion constructs (Nl, N2, etc.) used in FIGS. 6B and 7A (SEQ ID NO:12 through SEQ ID NO:15).
- WT indicates the full-length construct (SEQ ID NO:l). Regions of ammo acid residues removed from deletion constructs Dl and D2 (SEQ ID NO: 16 and SEQ ID NO: 17) are also shown.
- FIG. 2 shows a filter ⁇ -galactosidase assay demonstrating interaction of PDE4D5 with the receptor for activated protein C kmase (RACKl) , but not with the related beta-prime coatomer protein ( ⁇ '-COP) .
- the PDE4D5 cDNA was cloned into pLEXAN to produce fusions with the LexA DNA-bindmg domain.
- Various WD- repeat proteins were cloned into pGADN to produce fusions with the GAL4 activation domain.
- cerevisiae cells containing the appropriate plasmids were patched onto plates that selected for both plasmids and subjected to a filter ⁇ - galactosidase assay, as described m G.B. Bolger, in Protein Targeting Protocols 101-131 (R.A. Clegg ed. 1998) . Positive results in the assay produce a change m the color of the patches from pink to blue.
- the bottom two patches serve as internal positive and negative standards, respectively (the oncoprotems RAS v12 and RAF (A.B. Vo-jtek et al . , 74 Cell 205-214 (1993) ) , and the vectors without inserts) .
- FIGS. 3A and 3B show lmmunoblots of extracts of untransfected COS7 cells probed with PDE4D-spec ⁇ fIC antibody (FIG. 3A) or RACKl-specific antibody (FIG. 3B) .
- FIG. 3A extracts from untransfected COS7 cells were fractionated (see Experimental Procedures) and then immunoblotted with the PDE4D antibody.
- FIG. 3C shows an immunoblot of cytosolic fractions from untransfected COS7 cells subjected to immunoprecipitation with PDE4D-specific antibody (lanes marked “4D”) or RACKl-specific antibody (lanes marked “RACKl”); the immunoprecipitates were then separated by SDS polyacrylamide gel electrophoresis, and the upper half of the gel was immunoblotted with the PDE4D- specific antibody and the lower half with the RACKl-specific antibody.
- Cytosolic (S2) fractions were prepared from untransfected COS7 cells and either immunoblotted directly (lanes marked “none") , or subjected to immunoprecipitation with either the PDE4D antibody (lanes marked “4D”) or the RACKl antibody (lanes marked “RACKl”) .
- the fractions/immunoprecipitates were then separated by SDS-PAGE.
- the upper half of the gel was immunoblotted with the PDE4D- specific antibody.
- the arrows indicate the location of PDE4D3 and PDE4D5 as 95 and 105 kDa species, respectively.
- the lower half of the gel was immunoblotted with the RACKl-specific antibody, and the position of the 36 kDa RACKl species is indicated.
- FIGS. 3D-F show i munoblots of the combined pellet (lanes marked “p") and S2 (lanes marked “s") fractions of untransfected COS7 cells treated with 10 m-M phorbol-12- myristate-13-acetate (PMA) ; immunoblots were probed with protein kinase C (PKC) specific antibody (FIG. 3D), RACKl- specific antibody (FIG. 3E) , or PDE4D-specific antibody (FIG. 3F) . Cells were harvested at the indicated times (in minutes), fractionated as in FIGS. 3A-C and subjected to SDS- PAGE, followed by immunoblotting for PKC (FIG. 3D), RACKl (FIG.
- PKC protein kinase C
- PDE4D PDE4D
- FIG. 3F PDE4D
- All data are typical of experiments done at least three times. Each lane on the gels represents 50 ⁇ g of protein, with PDE activities in the range of 25-35 pmol/min/mg protein. Equal amounts of protein from the PI, P2, and S2 fractions were analyzed.
- the PDE4D antibodies did not immunoprecipitate purified GST-RACK1. The RACKl antibody did not immunoprecipitate purified MBP-PDE4D5 (data not shown) .
- FIGS. 4A and 4B show immunoblots of cytosolic extracts of Jurkat (FIG. 4A) , HEK-293, 3T3-F442A, and SK-N-SH cell lines (FIG. 4B) either immunoblotted directly (lanes marked “T") or subjected to immunoprecipitation with the RACKl-specific antibody (lanes marked “rIP” or “i”) or a non-specific mouse antiserum (lanes marked “n”); the upper half of the gel was immunoblotted with PDE4D-specific antibody, and the lower half of the gel was immunoblotted with RACKl-specific antibody.
- Cytosolic extracts were prepared from the Jurkat, HEK-293, 3T3-F442A and SK-N-SH cell lines. They were either immunoblotted directly (lanes marked “T”), or subjected to immunoprecipitation with the RACKl antibody (lanes marked
- RACKl was also co-immunoprecipitated with PDE4D5 when the initial immunoprecipitation was performed with the PDE4D5-specific antibody, as demonstrated by immunoblotting of PDE4D5 immunoprecipitates with the RACKl antibody (data not shown) .
- FIGS. 5A and 5B show SDS polyacrylamide gel electrophoresis separations of glutathione-S-transferase (GST) and a fusion between GST and RACKl (GST-RACKl) (FIG. 5A) ; and fusion between maltose-binding protein (MBP) and PDE4D3 (MBP- 4D3) or PDE4D5 (MBP-4D5) (FIG. 5B) . Fusions between GST and RACKl, and also between MBP and PDE4D3 or PDE4D5, were expressed and purified from E. coli (see Experimental Procedures). GST alone (i.e., not as a fusion) was expressed and purified in an identical manner.
- GST alone i.e., not as a fusion
- FIG. 5C shows the interaction of E.
- FIG. 5D shows dose-response curves calculated for the inhibition of PDE4D5 by rolipram at a concentration of substrate (cAMP) of 1.0 ⁇ M .
- Assays were performed on MBP- PDE4D5 ("4D5-MBP") alone, and also on MBP-PDE4D5 complexed with GST-RACKl. Assays were performed using an excess of GST- RACKl so that all of the PDE4D5 would be complexed with RACKl (see Experimental Procedures) . In pull-down experiments, all of the PDE4D5 could be shown to complex with GST-RACKl under these conditions (data not shown) . As a control, assays were performed with GST alone, added at comparable levels.
- FIGS. 6A-C show pull-down assays of recombinant forms of PDE4D5, i.e. wild-type recombinant PDE4D5 isoforms (FIG. 6A) and deletion mutants of PDE4D5 (FIGS. 6B and 6C) , with RACKl.
- various PDE4D isoforms (or mutant forms thereof) were expressed in C0S7 cells. Cytosolic extracts were prepared from the cells, mixed with GST-RACKl, and then subjected to affinity absorption on glutathione agarose beads, followed by SDS-PAGE and immunoblotting.
- FIG. 6A shows a pull-down experiment performed with wild-type recombinant PDE4D isoforms, wherein PDE4D3 or PDE4D5 was expressed in COS7 cells, and then subjected to "pull-downs" with GST-RACKl (lanes marked “rg") or GST alone (lanes marked “g”). After affinity absorption, the material on the beads was immunoblotted with the PDE4D antibody. Cytosolic extracts from the cells (i.e., not subjected to "pull-downs”) were run as standards (lanes marked "ly”) . In cells transfected with vector alone (“mock”), GST-RACKl pulled down a single 105 kDa species consistent with PDE4D5.
- FIG. 6B shows a pull-down experiment performed with constructs encoding deletions in PDE4D5.
- the regions of PDE4D5 included in the various constructs are shown in FIG. 1.
- FIG. 6B shows co-immunoprecipitations of RACKl and PDE4D5 mutants. Constructs encoding mutants of PDE4D5 with
- FIGS. 7A and 7B show yeast two-hybrid analysis of PDE4D5 deletions and point mutations.
- plasmids encoding fusions between the DNA-binding domain of LexA and various amino-terminal deletions of PDE4D5 were tested for their ability to interact with RACKl, expressed as a fusion with the GAL4 activation domain (right column, "pGADN-RACKl") .
- the identical LexA fusions were tested for their ability to interact with the GAL4 activation domain alone (left column, "pGADN”) .
- the regions of PDE4D5 included in the various constructs are shown in FIG. 1.
- ORF means open reading frame
- WD-repeat means a protein sequence motif containing repetitive sequences bound by tryptophan
- SD means standard deviation.
- 5. cerevisiae means Sa ccharomyces cerevisiae .
- single-letter code and similar terms refer to single-letter designations for the 20 ammo acid residues found in peptides and proteins, as follows: A - alanme, C - cysteme, D - aspartic acid, E - glutamic acid, F
- pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as dizziness and the like, when administered to a human.
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
- Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
- effective amount means an amount of a drug or pharmacologically active agent that is nontoxic but sufficient to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio attending any medical treatment.
- An effective amount of a peptide for inhibiting the interaction between PDE4D5 and RACKl is an amount sufficient to inhibit the interaction to a selected level .
- administering and similar terms mean delivering the composition to the individual being treated such that the composition is capable of being circulated systemically to the parts of the body where the peptide can inhibit the interaction between PDE4D5 and RACKl.
- the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration.
- injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
- Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added.
- Regulation of the levels of the second messenger cAMP has the potential to influence multiple processes in cells and tissues. PDE4 enzymes have been implicated in numerous cellular functions in the brain, airway smooth muscle, endocrine tissues, and in the immune/inflammatory systems (for review see M.D. Houslay et al . , 44 Advances in Pharmacology 225-342 (1998)). However, the regulation of many PDE4 isoforms is poorly understood.
- PDE4D5 A novel PDE4 isoform, PDE4D5, which is expressed in numerous cell lines and also in the brain, G.B. Bolger et al . , 328 Biochem J. 539-548 (1997), has recently been isolated. It is demonstrated herein that PDE4D5 interacts specifically and at high affinity with the RACKl WD- repeat "scaffold" protein. The interaction between these two proteins is demonstrated by multiple independent methods, including two-hybrid screening, pull-down assays with recombinant RACKl, and binding studies with purified recombinant proteins. It is also demonstrated by co- immunoprecipitation studies that native, endogenously expressed RACKl and PDE4D5 interact in cells.
- RACKl means Receptor for Activated Protein C Kinase.
- the first identified functional role for RACKl was as a protein capable of binding to various PKC isoforms after they had been activated through treatment of cells with either diacylglycerol or phorbol esters.
- the coatomer protein, ⁇ '-COP is also a WD-repeat protein that also interacts with PKC isoforms. M. Csukai et al . , 272 J. Biol. Chem. 29200-29206 (1997). It is shown herein that PDE4D5 does not bind to either G s ⁇ or ⁇ '-COP, demonstrating that PDE4D5 interacts specifically with RACKl and not with WD-repeat proteins in general.
- RACKl has been shown to interact with the integrin ⁇ subunit, J. Liliental & D.D. Chang, 273 J. Biol. Chem. 2379-2383 (1998), and the protein tyrosyl kinase Src, ; B.Y. Chang et al . , 18 Mol . Cell. Biol. 3245-3256 (1998).
- RACKl may serve as a "scaffold" or "adaptor” protein for either Src or the integrin ⁇ subunit.
- Overexpression of RACKl inhibits the tyrosine kinase activity of Src and inhibits the growth of NIH-3T3 cells. B.Y. Chang et al . , 18 Mol. Cell. Biol. 3245-
- RACKl The interaction of RACKl with the ⁇ subunit of integrins also requires the stimulation of cells with PMA. J. Liliental & D.D. Chang, 273 J. Biol. Chem.. 2379-2383 (1998). This suggests that PKC activation is necessary for the interaction of the integrin ⁇ subunit with RACKl, or that PMA can directly promote the interaction. This interaction may involve a conformational change in RACKl, as the interaction between RACKl and the integrin ⁇ subunit can only be demonstrated in vitro if a truncated form (WD-repeats 5 through 7, inclusive) of RACKl is used.
- PDE4 isoforms have also been demonstrated to bind to other signaling proteins, and it is of interest that these interactions also appear to be mediated by sequences m the unique ammo-termmal ends of the PDE4 proteins.
- the PDE4A4/5 isoform has been demonstrated to bind to proteins containing SH3 domains. J.C. O'Connell et al., 318 Biochem. J. 255-262 (1996) .
- the PDE4A1 isoform is targeted to membranes, and the membrane targeting of this isoform is mediated by specific ammo acids in its ammo- termmal region. I. McPhee et al . , 310 Biochem. J. 965-974 (1995); Y.
- the physiologic implications of the RACKl :PDE4D5 interaction may be related to the ability of RACKl to serve as a "scaffold” or "adaptor” protein that mediates the recruitment of PDE4D5 into a protein complex.
- a single "scaffold” or “adaptor” protein may interact with multiple different proteins, all of which can potentially be recruited into the complex.
- AKAPs A-kmase anchoring proteins
- PKC protein phosphatase 1.
- “Scaffold, " “anchor, “ and “adaptor” proteins physically connect various signal transduction components, such as receptors, kmases, and elements of the cytoskeleton, into stable complexes. These complexes bring enzymes closer to their regulatory components or substrates, or closer to other components of a signaling network. T. Pawson & J.D. Scott, 278 Science 2075-2080 (1997) . Therefore, the multidomam protein RACKl may serve as a "scaffold” able to recruit a variety of signal transduction proteins.
- RACKl ' s partners such as PKC isoforms and mtegrms
- PKC isoforms and mtegrms may be recruited to the complex only upon activation by phorbol esters
- others such as PDE4D5 and Src
- PDE4D5 and Src may be constitutively associated with RACKl in those cell types where both proteins are expressed.
- the functional significance of the association of PDE4D5 with RACKl remains to be elucidated.
- the recruitment of PDE4D5 to a signaling complex may provide a potential mechanism for the modulation of cAMP levels in the vicinity of the complex. This could turn regulate the activity of protein kinase A, which could influence the activity or function (s) of the complex, or of other adjacent cellular components.
- pLEXAN is a derivative of pBTMll ⁇ , P.L. Bartel & S. Fields, 254 Methods Enzymol. 241-263 (1995), with a o l site inserted into the polylinker. Screens were performed with the HeLa two-hybrid cDNA library in the S. cerevisiae strain L40. A.B. Vojtek et al., 74 Cell 205-214 (1993). To screen the HeLa cDNA library, positive clones were initially selected for growth in the absence of histidine (without 3-amino-triazole) , and then transferred to patches and assayed for LacZ activity using a filter ⁇ -galactosidase assay, as described in G.B.
- pGADN is a derivative of pGADGH, but with a Notl site inserted into the polylinker.
- Generation of bacterial expression constructs The full ORFs of PDE4D5, PDE4D3 and RACKl were cloned into the Notl site of pMALN, to generate pMALPDE4D5 (also called pMALP79) and pMALPDE4D3 (also called pMALN43), respectively.
- pMALN is a derivative of pMALc2 (New England Biolabs, Beverly, Massachusetts; C. Guan et al . , 67 21-30 (1988)), with a Notl site inserted into the polylinker.
- the full ORF of RACKl was cloned into the Notl site of pGEX-5X-3 (Pharmacia, Piscataway, New Jersey; D.B. Smith & K.S. Johnson, 67 Gene 31-40 (1988)) to generate pGEXRACKl .
- All constructs named "pMAL . . .” generate fusions between the maltose-binding protein (MBP) and the amino-terminus of the protein encoded by the insert.
- Constructs named "pGEX . . .” constructs generate fusions between glutathione-S-transferase (GST) and the amino-terminus of the protein encoded by the insert.
- VSV vesicular stomatitis virus glycoprotein epitope
- Verification of two-hybrid, expression and mutagenesis constructs All PCR-generated or mutant constructs were verified by sequencing prior to use. Growth of cell lines- All cell lines used in these experiments were obtained from the American Type Culture Collection (Manassas, Virginia) . The lines were grown in Dulbecco's modified Eagle's medium, supplemented with fetal calf serum and antibiotics.
- Co-immunoprecipita tions- COS7, SK-N-SH, Jurkat, 3T3-F442A or HEK 293 cells were harvested in 0.5 ml lysis buffer (55 mM Tris-HCl, pH 7.4, 132 mM NaCl, 22 mM sodium fluoride, 11 mM sodium pyrophosphate, 1.1 mM EDTA, 5.5 mM EGTA) containing complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, Indiana) and lysed with 8 strokes of a 26- ⁇ gauge needle attached to a disposable syringe. This method was used to permit processing of a large number of samples quickly.
- coli JM109 containing pGEXRACKl, pMALPDE4D3 or pMALPDE4D5 were induced with lm-M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG; Boehringer Mannheim, Indianapolis, Indiana) for 4 hours at 30°C.
- Bacteria were harvested by centrifugation at 2,500 x g for 10 minutes at 4°C, and the bacterial pellet was frozen at -80°C overnight.
- the bacterial pellets were resuspended in 10 ml of ice-cold resuspension buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 10 mM ⁇ -mercaptoethanol, and complete protease inhibitor cocktail) and sonicated with 4 x 30 sec bursts at maximal setting.
- Triton X-100 was added to a final concentration of 0.02% and cell debris was then removed by centrifugation at 15,000 x g for 10 minutes at 4°C.
- the cleared supernatant was incubated with 0.1 volume of pre- equilibrated glutathione sepharose beads (for GST fusions) or amylose resin (for MBP fusions) on an orbital shaker for 30 minutes at 4°C.
- the beads were collected by centrifugation at 2,000 x g for 1 minute and washed three times with ice-cold resuspension buffer.
- the fusion proteins were eluted by the addition of 5 mM glutathione, 50 mM Tris-HCl, pH 8.0 (for GST fusions) or 10 mM maltose, 50 mM Tris-HCl, pH 8.0 (for MBP fusions) and dialyzed three times against 50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5% glycerol.
- the purified fusion proteins were stored at -80°C until required.
- ELISA Protein Intera ction Assay- Reacti-Bind glutathione coated ELISA plates (Pierce, Rockford, Illinois) were treated with 1 g of purified GST or GST-RACKl for 16 hours at 4°C and then washed three times with 100 ⁇ l/well of wash buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). Dilutions of the MBP fusions of PDE4D3 and PDE4D5 were incubated with immobilized GST or GST-RACKl for 3 hours at room temperature and the wells were then washed three times with 100 ⁇ l/well of ice-cold wash buffer.
- Protein complexes were fixed with the addition of 100 ⁇ l/well 4% (v/v) paraformaldehyde in PBS for 30 minutes at 4°C. Paraformaldehyde fixation was used at this step because of concern that the complexes might dissociate during the subsequent detection procedure. However, it was determined subsequently that the binding of PDE4D5 and RACKl was so avid that the addition of paraformaldehyde made little difference.
- protein complex formation was detected by the addition of anti-PDE4D monoclonal antibody (1:10,000 (v/v) in dilution buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl] ) for 1 hour at room temperature, followed by alkaline phosphatase conjugated anti-mouse IgG (Sigma Chemical Co., St. Louis, Illinois; 1:2000 (v/v) in dilution buffer) for a further hour at room temperature.
- dilution buffer 10 mM Tris-HCl, pH 7.4, 150 mM NaCl
- Rolipram was dissolved in 100% DMSO as a 1 mM stock and diluted in 20 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 to provide a range of concentrations in the assay. The residual levels of DMSO were shown not to affect PDE activity over the ranges used in this study.
- Rvalues data from PDE assays were analyzed by computer fitting to the hyperbolic form of the Michaelis-Menten equation using an iterative least squares procedure (Ultrafit; with Marquardt algorithm, robust fit, experimental errors supplied; Biosoft, Ferguson, Missouri) . Relative V max values could be calculated using the Michaelis equation and the experimentally derived K m values, as described previously. Y. Shakur et al., 306 Biochem. J. 801- 809 (1995) .
- PDE4D5 To determine more about the properties of PDE4D5, it was determined to investigate whether specific proteins might bind to it. For this purpose, full-length PDE4D5 was used as a
- PDE4D5 was tested for its ability to bind to these proteins expressed as GAL4 fusions, and also to the GAL4 activation domain itself (i.e., not as a fusion) . No interaction was detected under conditions where an interaction could be shown between PDE4D5 and RACKl (data not shown) .
- PDE4D5 was also tested for its ability to bind to two other WD-repeat proteins, the G-protein G s ⁇ subunit and the coatomer subunit protein, ⁇ '-COP.
- the rationale for testing ⁇ '-COP was that, like RACKl, it has also been shown to bind to PKC isoforms, although with selectivity for different PKC isoforms than RACKl. M.
- COS7 cells were disrupted and fractionated to yield a high speed (S2) supernatant fraction reflecting cytosolic components, and also PI and P2 particulate fractions (see Experimental Procedures) .
- the fractions were subjected to SDS-PAGE and immunoblotted with antibodies specific for either PDE4D or RACKl. Both specific polyclonal and monoclonal PDE4D antibodies were used, with similar results.
- G.B. Bolger et al . , 328 Biochem J. 539-548 (1997) The PDE4D antibodies were all generated to a carboxy- terminal region of the PDE4D protein and have been shown to detect all five PDE4D species, G.B. Bolger et al . , 328 Biochem J. 539-548 (1997), as the PDE4D isoforms differ solely by their distinct amino-terminal regions (FIG. 1).
- PDE4D5 and RACKl can be co-immunoprecipi ta ted from COS7 cells-
- the cytosolic (S2) fraction from C0S7 cells was subjected to an immunoprecipitation protocol using either the RACKl antibody (FIG. 3C; lanes marked “RACKl”) or the PDE4D antibody (FIG. 3C; lanes marked "4D”).
- the resulting immunoprecipitates were then subjected to SDS- PAGE and immunoblotting. Since PDE4D5 migrates as a 105 kDa protein, G.B. Bolger et al . , 328 Biochem J.
- PDE4D antibody not only immunoprecipitated PDE4D3 and PDE4D5 but it also co-immunoprecipitated RACKl.
- PDE4D5 and RACKl can be co-immunoprecipi ta ted from various cell types-
- RACKl interaction occurred in cells generally, a number of other cell lines were examined, specifically HEK293 (human embryonic kidney), 3T3-F442A (mouse fibroblast/pre-adipocyte) , SK-N-SH (human neuroblastoma) , and Jurkat (human T-cell) . Cytosolic extracts from these cell lines were subjected to SDS-PAGE with subsequent immunoblotting. The top half of the immunoblot was treated with a PDE4D antibody and the bottom half with the RACKl antibody (FIGS.
- the data support the concept that PDE4D3 does not interact with RACKl, since PDE4D3 was not immunoprecipitated from HEK293 and 3T3-F442A cells using the RACKl antibody (FIG. 4B) .
- PDE4D5 and RACKl interact directly in vitro in a dose- dependent manner-
- RACKl and PDE4D5 interact directly in vitro in a dose- dependent manner-
- PDE4D5 was expressed as a maltose-binding protein fusion (MBP-PDE4D5) in E. coli . It was then purified (FIG.
- PDE4D3 as a purified MBP fusion protein (FIG. 5B) , was unable to bind to RACKl when the capture assay was performed under conditions identical to those that demonstrated interaction between RACKl and PDE4D5 (FIG. 5C) . This is consistent with the lack of interaction between PDE4D3 and RACKl in intact cells (FIGS. 3C and 4B) .
- Enzyma tic activi ty of RACKl -bound PDE4D5- To evaluate whether interaction with RACKl altered the catalytic activity of PDE4D5, the effect on PDE4D5 activity of complexing E. coli-purified recombinant PDE4D5 with E.
- RACKl interacts with PDE4D5, but not wi th other PDE4 isoforms- Five PDE4D isoforms have been identified to date (G.B. Bolger et al., 328 Biochem J. 539-548 (1997); FIG. 1). It has already been demonstrated herein that RACKl does not interact with PDE4D3 (FIGS. 3C, 4B, 5C) . To determine whether any of the PDE4D1, PDE4D2, PDE4D3, or PDE4D4 isoforms interact with RACKl, cDNAs encoding these isoforms were expressed in S.
- PDE4D5 The interaction of PDE4D5 with RACKl is media ted by specific amino acids in the PDE4D5 amino-terminal region- Since RACKl interacts specifically with PDE4D5, and not with other PDE4 isoforms, it is likely that RACKl interacts with regions of sequence that are unique to PDE4D5.
- PDE4D5 differs from all known PDE4 isoforms, M.D. Houslay et al . , 44 Advances in Pharmacology 225-342 (1998); G.B. Bolger et al., 328 Biochem J. 539-548 (1997) , in the presence of a unique region of 88 amino acids at its amino-terminus (FIG. 1; SEQ ID N0:1).
- pcDNA4D5VSV DNA template was added to STP3 Transcription Mix (10 ⁇ l total reaction volume) and incubated at 30°C for 15 minutes. Following the transcription step, 30 ⁇ l of STP3 Translation Mix and 30 ⁇ Ci of [ s 35] -methionine were added to the reaction tube. The reaction volume was made to 50 ⁇ l with nuclease-free water, and then the tube was incubated at 30°C for 60 minutes. This product was then used in binding experiments with GST-RACKl.
- Example 2 Two phage libraries used for library screening were the M13-phage-based heptapeptide (Ph.D. -7) and dodecapeptide (Ph.D. -12) random display libraries supplied by New England
- the PEG precipitate was collected by centrifugation (10, 000xg max , 10 minutes, 4°C) and resuspended in 1 ml TBS. Residual bacterial cells were removed by centrifugation at 13,000xg max for 5 minutes at 4°C and then the supernatant re-precipitated for 60 minutes at 4°C with 1/6 volume PEG/NaCl . The amplified eluate was collected by centrifugation (13, 000xg max , 10 minutes, 4°C) and resuspended in 200 ⁇ l TBS, 0.02% NaN 3 .
- the amplified phage pool was then pre- cleared, added to GST or GST-fusion protein coated wells, incubated, eluted and amplified as described above except that wash steps carried out with TBS + 0.5% (v/v) Tween 20. Four cycles of affinity enrichment were carried out for the Ph.D.- 12 library and three rounds of enrichment for the Ph.D. -7 library.
- Phage titers (of primary and amplified libraries, eluted and amplified phage) were determined following infection of 200 ⁇ l of mid-log phase E. coli ER2537 with 10 ⁇ l of LB- diluted phage for 5 minutes at room temperature.
- Infected cells were then transferred to a culture tube containing 3 ml of pre-warmed (45°C) Agarose Top (10 g/1 (w/v) tryptone, 5 g/1 (w/v) yeast extract, 5 g/1 (w/v) NaCl, 1 g/1 MgCl 2 .6H 2 0, 7 g/1 agarose) and immediately poured onto pre-warmed (37°C) LB/isopropyl ⁇ -D-thiogalactoside (IPTG) /5-bromo-4-chloro-3- indolyl- ⁇ -D-galactoside (Xgal) plates (LB medium containing 15 mg/ml (w/v) agar, 80 ⁇ g/ml (w/v) IPTG, 3 ⁇ g/ml (w/v) Xgal) .
- IPTG ⁇ -D-thiogalactoside
- Xgal indolyl- ⁇ -D-galactoside
- phage-containing pellet resuspended in 100 ⁇ l iodide buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4 M Nal).
- iodide buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4 M Nal.
- Preferential precipitation of single-stranded phage DNA was accomplished by the addition of 250 ⁇ l of ethanol followed by incubation for 10 minutes at room temperature.
- Precipitated DNA was collected by centrifugation (13, 000xg max , 10 minutes, 4°C) , washed with 70% (v/v) ethanol/H 2 0 and dried under vacuum.
- DNA sequencing of recombinant phage inserts was done by the dideoxy chain termination technique using the fluorescent dye mixture for automated sequencing (ABI) and the -96gIII sequencing primer supplied by New England Biolabs.
- TE buffer 10 mM Tris/HCl, pH 8.0, 1 mM EDTA.
- DNA sequencing of recombinant phage inserts was done by the dideoxy chain termination technique using the fluorescent dye mixture for automated sequencing (ABI) and the -96gIII sequencing primer supplied by New England Biolabs.
- ABSI fluorescent dye mixture for automated sequencing
- SEQ ID NO: 19 25mer peptide
- ELISA plate wells were coated with peptide (10 ⁇ g) or selected phage clones (1 x 10 12 virions) diluted in 200 ⁇ l of 0.1 M NaHC0 3 (pH 8.6) and incubated overnight at 4°C in an air-tight humidified container. Plates were then blocked for 2 hours at 4°C with 200 ⁇ l/well of 5 mg/ml BSA in 0.1 M NaHC0 3 (pH 8.6) .
- Wells were washed 6 times with TBST (TBS + 0.5% (v/v) Tween- 20) and then incubated for 1 hour at room temperature with GST-RACKl (in a range of concentrations of 2 mM to 50 pM) diluted in TBST) and then the wells were washed 6 times with ice cold TBST.
- Wells were then washed 6 times with ice cold TBST.
- HRP horse radish peroxidase
- goat anti-mouse IgM goat anti-mouse IgM
- phage-based pill-protein display libraries were screened against GST, GST-RACKl and GST-LYN-SH3.
- One of these consisted of 7 random residues linked to the pVIII protein by a linker of three tandem copies of the sequence Gly 4 Ser.
- affinity selection and amplification were performed, whereas four rounds were performed for the 12-mer library.
- domain-specific phage libraries were incubated with immobilized GST or MBP prior to affinity selection.
- a phage-based ELISA was employed to assess the binding specificity of individual phage clones.
- the highest frequency of LYN-SH3-specific phage recovered contained the class I ligand consensus motif Arg-Pro-Leu-Pro- Pro-Leu-Pro (SEQ ID NO: 26) .
- the majority of GST- specific phage contained the sequence motif His-His-Ser-His- Thr Pro Arg (SEQ ID NO:21) demonstrating the specificity of the screening process for the LYN-SH3 fusion-partner protein and not for GST.
- RACKl phage did not contain any of the sequence characteristics of LYN-SH3- or GST-specific clones. However, no consistent sequence motifs were obtained from RACKl phage screened against the 7-mer library.
- Xaa is a hydrophobic amino acid (Pro or Ala) .
- Pro-Xaa-Leu core all RACKl phage were rich in other hydrophobic amino acids (Leu, lie, Ala, Phe, and Trp) , which were found carboxy- and amino-terminal to the Pro-Xaa- Leu motif.
- Other sequence characteristics of the collection of RACKl phage included a propensity for an acidic amino acid (Asp or Glu) , which only occurred amino terminal to the Pro- Xaa-Leu motif.
- RACKl-specific phage also contained a strongly basic lysine residue carboxy terminal to the Pro-Xaa-Leu motif.
- the organization of amino acids upstream of the proline and downstream of the lysine residues yielded the mixed consensus sequence Glu-Xaa-Pro-Xaa- Leu (SEQ ID NO:51) .
- Tables 2-6 show the amino acid sequences of the randomized regions of GST, LYN-SH3, and RACKl specific phage.
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EP00937642A EP1183391A2 (en) | 1999-05-20 | 2000-05-20 | Interaction of the cyclic-amp-specific phosphodiesterase pde4d5 with rack1 |
AU52786/00A AU5278600A (en) | 1999-05-20 | 2000-05-20 | Interaction of the cyclic-amp-specific phosphodiesterase pde4d5 with rack1 |
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