Binding of HTm4 to Cyclin-dependent Kinase (Cdk)-associated Phosphatase (KAP)·Cdk2·Cyclin A Complex Enhances the Phosphatase Activity of KAP, Dissociates Cyclin A, and Facilitates KAP Dephosphorylation of Cdk2

Abstract

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP·cdk2 complex. Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP·cdk2 complex. HTm4 (MS4A3) is the third member of subfamily A in an extensive membrane-spanning four-domain gene family. These genes are only loosely related at the sequence level, but their encoded proteins share a common four-transmembrane topology, including CD20 (MS4A1) and FcϵRI (MS4A2). To date, few functions for the MS4 family of proteins have been ascribed. However, a diverse functionality is beginning to emerge. These functions include roles such as cell surface signaling receptors and intracellular adapter proteins (1Ishibashi K. Suzuki M. Sasaki S. Imai M. Gene (Amst.). 2001; 264: 87-93Crossref PubMed Scopus (82) Google Scholar, 2Liang Y. Buckley T.R. Tu L. Langdon S.D. Tedder T.F. Immunogenetics. 2001; 53: 357-368Crossref PubMed Scopus (81) Google Scholar, 3Liang Y. Tedder T.F. Genomics. 2001; 72: 119-127Crossref PubMed Scopus (102) Google Scholar, 4Hulett M.D. Pagler E. Hornby J.R. Immunol. Cell Biol. 2001; 79: 345-349Crossref PubMed Scopus (6) Google Scholar, 5Barnhill J.C. Stokes A.J. Koblan-Huberson M. Shimoda L.M. Muraguchi A. Adra C.N. Turner H. J. Cell. Biochem. 2004; 91: 808-820Crossref PubMed Scopus (38) Google Scholar, 22Adra C.N. Lelias J.M. Kobayashi H. Kaghad M. Morrison P. Rowley J.D. Lim B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10178-10182Crossref PubMed Scopus (48) Google Scholar). Cell cycle progression is regulated by the sequential activation, and inactivation, of the cyclin-dependent kinases (cdks). 1The abbreviations used are: cdk, cyclin-dependent kinase; DTT, dithiothreitol; KAP, cdk-associated phosphatase; p-cdk2, phosphorylated cdk2; PDB, Protein Data Bank; pNPP, p-nitrophenyl phosphate; RU, resonance units. 1The abbreviations used are: cdk, cyclin-dependent kinase; DTT, dithiothreitol; KAP, cdk-associated phosphatase; p-cdk2, phosphorylated cdk2; PDB, Protein Data Bank; pNPP, p-nitrophenyl phosphate; RU, resonance units. Cdks, themselves, are substrates for regulatory phosphorylation and dephosphorylation. The phosphorylation status of cdks is controlled by a discrete class of regulatory kinases and phosphatases (6Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1787) Google Scholar). Cdk activity is controlled by activatory and inhibitory proteins, the latter exemplified by p21 and p27. Binding of inhibitors such as p21 and p27 to different cyclin·cdk complexes is sufficient to arrest the cell cycle (7Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5220) Google Scholar, 8Tsai L.H. Lees E. Faha B. Harlow E. Riabowol K. Oncogene. 1993; 8: 1593-1602PubMed Google Scholar, 9Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4954) Google Scholar, 10Steinman R.A. Huang J. Yaroslavskiy B. Goff J.P. Ball E.D. Nguyen A. Blood. 1998; 91: 4531-4542Crossref PubMed Google Scholar, 11Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5110) Google Scholar, 12Taniguchi T. Endo H. Chikatsu N. Uchimaru K. Asano S. Fujita T. Nakahata T. Motokura T. Blood. 1999; 93: 4167-4178Crossref PubMed Google Scholar). Conversely, inactive, monomeric cdk can be activated via association with a specific cyclin and the concurrent phosphorylation of a conserved and essential threonine residue, such as threonine 160 (Thr160) in cdk2, which is located within the activation segment (T-loop) of the kinase. In addition to cyclin association, full activation of cdk2 requires phosphorylation of Thr160 and dephosphorylation of Thr14 and Tyr15. Thr160 of cdk2 is phosphorylated by CAK (cdk-associated kinase), whereas its dephosphorylation is critical for inactivation. This dephosphorylation is executed by a serine/threonine-directed phosphatase, KAP. It has been shown that exogenous expression of KAP slows the G1 phase cell cycle progression in HeLa cells and that aberrant KAP transcripts are detected in some hepatocellular carcinomas (13Lee S.W. Reimer C.L. Fang L. Iruela-Arispe M.L. Aaronson S.A. Mol. Cell. Biol. 2000; 20: 1723-1732Crossref PubMed Scopus (69) Google Scholar, 14Yeh C.T. Lu S.C. Chao C.H. Chao M.L. Biochem. Biophys. Res. Commun. 2003; 305: 311-314Crossref PubMed Scopus (21) Google Scholar). These observations suggest that KAP has the same biological effects as cdk inhibitors, although their modes of actions are different. KAP can bind to cdk2 either in the presence or absence of cyclins (15Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar, 16Poon R.Y. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar, 17Hannon G.J. Casso D. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1731-1735Crossref PubMed Scopus (110) Google Scholar). However, KAP can only dephosphorylate cdk2 when cyclin A is degraded or disassociated, in a mechanism that may control access of KAP to its substrate Thr160 (16Poon R.Y. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar). Cyclin binding may therefore control access of KAP to its substrate phosphothreonine 160. We reported previously that HTm4 interacts directly with the KAP via its C terminus (18Donato J.L. Ko J. Kutok J.L. Cheng T. Shirakawa T. Mao X.Q. Beach D. Scadden D.T. Sayegh M.H. Adra C.N. J. Clin. Invest. 2002; 109: 51-58Crossref PubMed Scopus (59) Google Scholar, 19Kutok J.L. Yang X. Folkerth R.D. Imitola J. Raddassi K. Yano Y. Salahuddin S. Lawitts J. Imboden H. Chinami M. Shirakawa T. Turner H. Khoury S. Sayegh M.H. Scadden D.T. Adra C.N. J. Mol. Histology. 2004; 36: 77-87Crossref Scopus (7) Google Scholar) and that exogenous expression of HTm4 leads to dephosphorylation of cdk2 and cell cycle arrest at the G0/G1 phase. Here, we show that the presence of HTm4 in the KAP·cdk2·cyclin A complex controls cdk2 activity in a dual fashion. First, HTm4 binding causes exclusion of cyclin A from its interaction with cdk2. Second, HTm4 binding potentiates KAP enzymatic activity and causes conformational changes that regulate access to Thr160. Subcloning, Expression, Purification, and Characterization of HTm4 and KAP—Human HTm4 was reverse transcribed-PCR cloned from total RNA extracted from human peripheral blood mononuclear cells from healthy volunteers with informed consent, using the following 5′- and 3′-primers (forward primer: hHTm4BamH1: 5′-cgc-gga-tcc-ggc-tct-gga-tct-gga-tct-ggt-atc-gag-gga-agg-atg-gcc-tcc-cac-gaa-gtt-3′; reverse primer: hHTm4Pst1: 5′-gca-ctg-cag-tta-ttg-att-aca-cag-aat-tgg-3′). The PCR product was subcloned into the pQE-80L vector (Qiagen). An N-terminal His tag was conferred during subcloning. Recombinant HTm4 was purified from transformed JM109 Escherichia coli as follows. Protein production was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to bacterial liquid culture in logarithmic phase. Bacteria were harvested by centrifugation at 6,000 rpm for 30 min. The bacterial pellet was lysed by sonication on ice (20 KW, 15 min) in 50 ml of N buffer (20 mm Tris-HCl, pH 8.0, containing 0.1 m NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm β-mercaptoethanol). The bacterial lysate was centrifuged at 20 × 104 g at 4 °C for 30 min. The remaining pellet was lysed in 40 ml of N buffer containing 6 m guanidine HCl. Proteins were bound to a 20-ml nickel-chelated Sepharose column (Amersham Biosciences) and were refolded by exposure to a 6–0 m urea gradient at 0.2 ml/min. The refolded protein sample was eluted with a gradient of N buffer containing 0.5 m imidazole and 0.3% deoxycholate. The peak fraction containing HTm4 was collected and purified by XArrest agarose after His tag cleavage with 4 units of Factor Xa (Novagen). Wild-type KAP, inactive KAP (C140S), and mutant KAP were expressed and purified as described previously (20Song H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Western Blotting of HTm4 —For the Western blot, 10 μl of purified protein was boiled in SDS sample buffer and resolved by 15% PAGE. After electrotransfer to polyvinylidene difluoride, the membrane (Bio-Rad) was blocked followed by either incubation with either anti-His tag antibody diluted at 1:5,000 (MBL) or anti-HTm4 antibody (Zymed Laboratories Inc.) and horseradish peroxidase-conjugated anti-rabbit IgG. Signals were detected using ECL (Amersham Biosciences). Protein Interaction Analyses (Surface Plasmon Resonance Assay)— The immobilization of proteins to sensor chips (CM-5 chip) was performed using a carbodiimide covalent linkage protocol (Amersham Biosciences). KAP was to the sensor at resonance units human was to the surface of the sensor antibody at Protein interaction were using the An HTm4 of was HTm4 protein (20 or HTm4 in 50 mm containing 1 mm 1 mm DTT, and 20 was at a of 10 Protein phosphatase activity was by the of of p-nitrophenyl as described previously (20Song H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The of phosphatase activity for KAP was 20 μl of containing 50 mm Tris-HCl, pH m NaCl, 2 mm DTT, 0.5 mm mm pNPP, 40 KAP, 4 A and 20 HTm4 was at 30 °C for the The was by the addition of 0.1 m The phosphatase activity in μl of sample was as To the of HTm4 on KAP, KAP phosphatase activity was with different HTm4 in the presence of was in the presence of HTm4. 40 KAP and 4 A. of either or KAP were in the different with or 20 HTm4 at 0.1 or 4 A the of HTm4 on KAP, the of KAP was using different of A as the substrate in the presence or absence of HTm4. The were using a KAP of or Protein dephosphorylation of phosphorylated proteins or substrates was using the 1 to the Thr160 of cdk2 and were used as substrates for the of KAP and HTm4 were 20 of and substrates were 10 and were performed in containing 50 mm Tris-HCl, pH and To the of the HTm4 and to the KAP phosphatase activity of an HTm4 of was The phosphatase activity of the HTm4 was using the protocol described were performed in with the and on the 2 and KAP phosphatase of KAP phosphatase 20 μl of containing 50 mm Tris-HCl, pH m NaCl, 2 mm DTT, 0.5 mm mm pNPP, 40 KAP, 4 A, and 20 HTm4 was at 30 °C for the The was by the addition of 0.1 m The phosphatase activity in μl of sample was at the presence of HTm4 KAP phosphatase KAP phosphatase activity was in the presence of of HTm4 using as the KAP The was in the presence of HTm4. 40 KAP and 4 A. were in and are KAP phosphatase activity was using the 1 with different of A 4 to 20 as the substrate in the presence or absence of 40 HTm4. and for the in the presence and absence of HTm4 are and KAP phosphatase activity was using the 1 with different of A 4 to 20 as the substrate in the presence or absence of 40 HTm4. effects of HTm4 on KAP phosphatase activity of either or inactive mutant KAP were in the different with or 20 HTm4 at 0.1 or with 4 A effects of HTm4 on KAP phosphatase activity with phosphorylated cdk2 or as its threonine 160 from A, and were used as substrates to KAP were performed using the of KAP and HTm4 were 20 and of and were 10 and were in the which contains 50 mm Tris-HCl, pH and The was to for 30 at 30 and was at were performed on a were from to The of KAP was by different KAP in the same (20 mm Tris-HCl, pH m A KAP and an of HTm4 The of sample were to in sample A, for HTm4 The in was to and on the in Cyclin A status of the A complex was in the presence and absence of HTm4. Recombinant A and KAP were in vitro and in the absence or presence of either 1 or 10 HTm4. The were to a to KAP protein and binding The bound were resolved by and Western for the presence of cyclin A An phosphatase to anti-rabbit was used to the of bound cyclin A present in the KAP·cdk2 complex. 10 μl of 10 A and 10 μl of 10 KAP were in 20 mm Tris-HCl, pH buffer containing m NaCl, and After at for either 10 HTm4 His or its buffer was to the After at for μl of was to The bound to KAP were with the buffer and boiled for The was with a Binding of KAP and HTm4 in have shown previously that HTm4 and KAP phosphatase may be from cells (18Donato J.L. Ko J. Kutok J.L. Cheng T. Shirakawa T. Mao X.Q. Beach D. Scadden D.T. Sayegh M.H. Adra C.N. J. Clin. Invest. 2002; 109: 51-58Crossref PubMed Scopus (59) Google Scholar). data the for a HTm4 is a of the cell cycle is that we the interaction is direct or an We have a purified interaction that to HTm4 and KAP HTm4 was in E. coli and purified by protein liquid The eluted peak to HTm4 was by and by Western anti-HTm4 and anti-His detected a to HTm4 and resonance was used to direct binding of purified HTm4 to KAP. KAP protein was to a sensor (Amersham Biosciences). with either or chips to antibody an in was when either HTm4 or a from HTm4 was sensor HTm4 with KAP the binding not These in vitro data that HTm4 and KAP directly and that the HTm4 C terminus is and sufficient to binding to KAP. KAP in the of HTm4 is a direct binding for KAP, as shown in We that HTm4 may regulate the activity of the KAP In the KAP phosphatase activity was using as a substrate for KAP. In the presence of HTm4 and the a for KAP activity was shown in after the addition of substrate, an phase the 20 min) was followed by a phase 30 The presence of HTm4 the phosphatase activity of KAP the and for KAP phosphatase activity were and and However, when HTm4 was the of KAP phosphatase activity to A in was also data suggest that the presence of HTm4 directly the activity of the KAP KAP of or Protein KAP dephosphorylation of phosphorylated proteins or substrates was using the 1 to the Thr160 of cdk2 and were used as substrates for the of KAP and HTm4 were 20 of and substrates were 10 and were performed in containing 50 mm Tris-HCl, pH and To the of the HTm4 and to the KAP phosphatase activity of a containing the of the HTm4 C terminus was The phosphatase activity of the HTm4 was using the protocol described were performed in with the and on the We the KAP phosphatase we have KAP phosphatase activity is in the presence of HTm4. an inactive KAP mutant is for the KAP, the phosphatase activity is The inactive KAP mutant as a control (15Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar, H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We that when HTm4 was or was with a KAP phosphatase activity was with control containing KAP, and A complex It is to that a A either a monomeric cdk2 or cyclin A, was for a KAP phosphatase activity induced by HTm4. data suggest that KAP activity is in the of a protein complex that contains its substrate, cdk2, and the HTm4 data that KAP and HTm4 are essential for KAP phosphatase on and we to the of was sufficient to the interaction and to the KAP phosphatase To this we the same containing the KAP binding as described into in vitro KAP activity the HTm4 a on KAP phosphatase activity with HTm4 protein These data that the C terminus of HTm4 direct binding of HTm4 to KAP and show that the C terminus is the of HTm4 which KAP HTm4 the of KAP data that HTm4 the activity of the KAP phosphatase a substrate We HTm4 also the activity of KAP its We the of KAP to dephosphorylate of its substrates First, we cdk2 with a phosphorylated Thr160 which as a substrate for KAP. We used a threonine We that HTm4 the activity of KAP phosphorylated cdk2 by when cdk2 is in the of the A complex. We also that HTm4 potentiates dephosphorylation of the However, KAP dephosphorylation activity be detected HTm4 was not dephosphorylation was when phosphorylated cdk2 was in the complex. These data that the presence of HTm4 a of KAP phosphatase of KAP by of HTm4 on that of HTm4 protein KAP we that the binding of HTm4 to the KAP induced KAP conformational To this we conformational changes of KAP in the presence or absence of the by using The of a protein in the can be to of the are the and and the are to the of the Signals in the from to are to from to are to and from to are to to the shown in we a at in the of KAP between sample A HTm4 and sample The a conformational in when HTm4 was into KAP HTm4 has this in the be by conformational changes in the KAP this that HTm4 direct in KAP after its binding to KAP by the C HTm4 Cyclin A from the activity of cdk2 is controlled by its with KAP and cyclin A. The binding of KAP phosphatase to the A complex and cdk2. However, the binding of cyclin A the of KAP to dephosphorylate cdk2. of cyclin A by HTm4 facilitates cdk2 dephosphorylation by KAP. HTm4 the KAP dephosphorylation on phosphorylated cdk2 that is in a complex with cyclin A. This that HTm4 may cdk2 by the inhibitory of cyclin A. that HTm4 may regulate cdk2 via KAP, and by cyclin A from its inhibitory interaction with cdk2. To we used an to the A interaction with and HTm4. We KAP protein using a binding cyclin A in the KAP·cdk2·cyclin complex However, when purified HTm4 His tag was to the KAP·cdk2·cyclin A cyclin A from the complex in a and the remaining protein complex was to only These observations that HTm4 cdk2 by KAP and by dissociation of cyclin A from cdk2. with the that HTm4 KAP phosphatase we suggest that HTm4 is as a in the activation of KAP and for cdk2 for of Cyclin A from was used to the of the HTm4 regulatory interaction that KAP phosphatase activity and leads to the exclusion of cyclin A. on we show that KAP conformational changes following binding to the HTm4 and that conformational changes may the interaction between the KAP substrate and its To this we a using the of A Biol. 1996; PubMed Scopus Google Scholar) and (20Song H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) from the Protein Data These were in The complex is shown in cdk and the is with the of the T-loop In this in cdk can be on are from the A or the complex is a as shown in We that the a site for the HTm4 C the reverse of the phosphothreonine 160 of cdk2 can be into the of the site of KAP in the absence of cyclin A, whereas a of substrate such as may be to the in the presence of cyclin A. This may KAP activity is at in the presence of the A complex. of of the T-loop in the and of HTm4 T-loop is a conserved segment in which an in cdk In the A phosphorylation is with the of the T-loop T. Endo H. Chikatsu N. Uchimaru K. Asano S. Fujita T. Nakahata T. Motokura T. Blood. 1999; 93: 4167-4178Crossref PubMed Google Scholar). However, cyclins can which to Thr160 phosphorylation in cdk2. The of interaction between the T-loop of and is shown to be critical the interaction between and was that of cdk2 and cyclin A (15Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1321) Google Scholar). This interaction is by the T-loop of the of the T-loop is critical for cdk In the A activation between and of the cdk2 T-loop and and of cyclin A (6Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1787) Google Scholar). However, in KAP·cdk2·cyclin A complex the A interaction is the T-loop is of the interaction of KAP This that binding of KAP to cdk2 the between cdk2 and cyclin A. is reported that between the T-loop of cdk2 and KAP between and of KAP with of cdk2 (20Song H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). the interaction between the T-loop of cdk2 and cyclin A, a specific interaction also between the at of cdk2 and the of of cyclin A in A complex. This interaction when the cyclin A is as in the complex the of the T-loop in A is by this In 20 of are for cdk2, with and cyclin A or its We of the T-loop and between and which are conserved in for cdk2 is with cyclin A, the T-loop is with at Conversely, cyclin A, the is with bound to KAP cyclin the T-loop of cdk2 in the to the of A, this at is to the be to an between and in the T-loop of cdk2 from the in a cyclin A from cdk2, the T-loop at the residue, and phosphothreonine 160 can be into the site of KAP. the of phosphothreonine 160 to KAP phosphatase in to HTm4 binding to KAP. in but not as shown in whereas its is conserved in cyclins A, and C but not in cyclin D. This that for cdk by KAP may between cdk and between different of the cell Binding of HTm4 we show the between Thr160 of cdk2 and of KAP, which that the of KAP the HTm4 C terminus via the of KAP. following HTm4 the conformational of the of the interaction between cdk2 and cyclin A and T-loop to and This HTm4 binding cyclin A from cdk2, a of cdk2. the reverse of the phosphothreonine 160 of cdk2 is into the of the site of KAP, after cyclin A is of for HTm4 has C.N. Lelias J.M. Kobayashi H. Kaghad M. Morrison P. Rowley J.D. Lim B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10178-10182Crossref PubMed Scopus (48) Google Scholar). this that are for Y. Buckley T.R. Tu L. Langdon S.D. Tedder T.F. Immunogenetics. 2001; 53: 357-368Crossref PubMed Scopus (81) Google Scholar). HTm4 to the nuclear We that the of HTm4 into the nuclear and the KAP·cdk2·cyclin A complex The interaction between HTm4 and the KAP·cdk2·cyclin A complex is and may only at in the cell HTm4 the activity of KAP, cdk2, and cyclin A. HTm4 also cdk2 activity in a dual by KAP activity and the of Thr160 to KAP by dissociation of inhibitory cyclin A In we show that HTm4 the cell cycle in cells its to control cdk2 We D. and K. of for K. of for column and T. and K. of for