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Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1

2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês

10.1074/jbc.m410317200

ISSN

1083-351X

Autores

Guisheng Zhou, Jonathan Boomer, Tse‐Hua Tan,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway. Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway. Hematopoietic progenitor kinase 1 (HPK1 1The abbreviations used are: HPK1, hematopoietic progenitor kinase-1; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; GCK, germinal center kinase; GLK, GCK-like kinase; PP4, protein phosphatase 4; PP2A, protein phosphatase 2A; TNF-α, tumor necrosis factor α; HA, hemagglutinin; LLnL, N-acetyl-l-leucyl-leucyl-l-norleucinal; MBP, myelin basic protein; HPK1-KD, the kinase domain of HPK1; HPK1-CD, the C-terminal regulatory domain of HPK1; HPK1-PR, the proline-rich region of HPK1; HPK1-DR, the distal region of HPK1; TCR, T cell receptor; HEK293T, human embryonic kidney 293T; DMEM, Dulbecco's modified Eagle's medium; Ab, antibody.; also named MAP4K1) belongs to the HPK1/GCk subgroup of mammalian Ste20-like kinases that specifically activate the JNK pathway and is considered as a potential MAPK kinase kinase kinase (1Chen Y.-R. Tan T.-H. Gene Ther. Mol. Biol. 1999; 4: 83-98Google Scholar, 2Chen Y.-R. Tan T.-H. Int. J. Oncol. 2000; 16: 651-662PubMed Google Scholar). The HPK1/GCk subgroup of kinases also includes germinal center kinase (GCK), GCK-like kinase (GLK), HPK/GCK-like kinase/NcK-interacting kinase, and kinase homologous to Ste20/Sps1/GCK-related (1Chen Y.-R. Tan T.-H. Gene Ther. Mol. Biol. 1999; 4: 83-98Google Scholar). These kinases are characterized by an N-terminal kinase domain, a C-terminal regulatory region, and the lack of a Rac1/Cdc42-binding domain found in p21-activated kinases, the other subgroup of mammalian Ste20-like kinases (1Chen Y.-R. Tan T.-H. Gene Ther. Mol. Biol. 1999; 4: 83-98Google Scholar, 3Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). HPK1 is unique in that its expression is restricted to hematopoietic tissues of adults, although it is widely expressed in embryos (4Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. 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Chem. 2001; 3: 45207-45216Abstract Full Text Full Text PDF Scopus (91) Google Scholar), and Grb2-related adaptor downstream of Shc (also called Grap 2) (13Liu S.K. Smith C.A. Arnold R. Kiefer F. McGlade C.J. J. Immunol. 2000; 165: 1417-1426Crossref PubMed Scopus (57) Google Scholar, 23Ma W. Xia C. Ling P. Qiu M. Tan T.-H. Liu M. Oncogene. 2001; 20: 1703-1714Crossref PubMed Scopus (44) Google Scholar, 24Lewitzky M. Harkiolaki M. Domart M.C. Jones E.Y. Feller S.M. J. Biol. Chem. 2004; 279: 28724-28732Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). HPK1 also constitutively interacts with a variety of adaptor proteins such as Grb2 (7Anafi M. Kiefer F. Gish G.D. Mbamalu G. Iscove N.N. Pawson T. J. Biol. Chem. 1997; 272: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 8Ling P. Yao Z. Meyer C.F. Wang X.S. Oehl W. Feller S.M. Tan T.-H. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar, 14Liou J. Kiefer F. Dang A. 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J. Biol. Chem. 1999; 274: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Le Bras S. Foucault I. Foussat A. Brignone C. Acuto O. Deckert M. J. Biol. Chem. 2004; 279: 15550-15560Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and Grap (14Liou J. Kiefer F. Dang A. Hashimoto A. Cobb M.H. Kurosaki T. Weiss A. Immunity. 2000; 12: 399-408Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We have shown that the adaptor protein, the HPK1-interacting protein of 55 kDa, is involved in the kinase activation of HPK1 by TCR stimulation (28Han J. Kori R. Shui J.W. Chen Y.R. Yao Z. Tan T.-H. J. Biol. Chem. 2003; 278: 52195-52202Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A recent phage display analysis found that HPK1 interacts with the cytoplasmic tail of membrane immunoglobulins (29Geisberger R. Prlic M. Achatz-Straussberger G. Oberndorfer I. Luger E. Lamers M. Crameri R. Appenzeller U. Wienands J. Breitenbach M. Ferreira F. Achatz G. Dev. Immunol. 2002; 9: 127-134Crossref PubMed Scopus (15) Google Scholar). Although phosphorylation (4Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar, 5Hu M. C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar, 7Anafi M. Kiefer F. Gish G.D. Mbamalu G. Iscove N.N. Pawson T. J. Biol. Chem. 1997; 272: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 15Ling P. Meyer C.F. Redmond L.P. Shui J.-W. Davis B. Rich R.R. Hu M. C.-T. Wange R.L. Tan T.-H. J. Biol. Chem. 2001; 276: 18908-18914Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-540Crossref PubMed Scopus (55) Google Scholar, 17Yu J. Riou C. Davidson D. Minhas R. Robson J.D. Julius M. Arnold R. Kiefer F. Veillette A. Mol. Cell. Biol. 2001; 21: 6102-6112Crossref PubMed Scopus (39) Google Scholar, 18Sauer K. Liou J. Singh S.B. Yablonski D. Weiss A. Perlmutter R.M. J. Biol. Chem. 2001; 3: 45207-45216Abstract Full Text Full Text PDF Scopus (91) Google Scholar, 30Ito Y. Pandey P. Sathyanarayana P. Ling P. Rana A. Weichselbaum R. Tan T.-H. Kufe D. Kharbanda S. J. Biol. Chem. 2001; 276: 18130-18138Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and caspase-mediated cleavage (19Chen Y.-R. Meyer C.F. Ahmed B. Yao Z. Tan T.-H. Oncogene. 1999; 18: 7370-7377Crossref PubMed Scopus (67) Google Scholar, 22Arnold R. Liou J. Drexler H.C. Weiss A. Kiefer F. J. Biol. Chem. 2001; 276: 14675-14684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have been implicated in the regulation of HPK1, the details of the in vivo regulation of HPK1 remain largely unknown. Protein phosphatase 4 (PP4; also called PPX and PPP4) is a protein serine/threonine phosphatase that is structurally related to the PP2A family of phosphatases (31Brewis N.D. Cohen P.T. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar, 32Hu M. C.-T. Shui J.-W. Mihindukulasuriya K.A. Tan T.-H. Gene (Amst.). 2001; 278: 89-99Crossref PubMed Scopus (16) Google Scholar). PP4 has been highly conserved over evolution, with human and Drosophila PP4 sharing 91% amino acid identity (31Brewis N.D. Cohen P.T. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar). Like PP2A, PP4 is a holoenzyme composed of catalytic (C), structural (A), and regulatory (B) subunits. To date, three PP4 subunits have been identified: α4, PP4-R1, and PP4-R2 (33Chen J. Peterson R.T. Schreiber S.L. Biochem. Biophys. Res. Commun. 1998; 247: 827-832Crossref PubMed Scopus (159) Google Scholar, 34Nanahoshi M. Tsujishita Y. Tokunaga C. Inui S. Sakaguchi N. 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Our previous studies show that PP4 interacts with components of NF-κB (e.g. c-Rel, p50, and RelA), stimulates the DNA binding activity of c-Rel, and activates NF-κB-mediated transcription (44Hu M. C.-T. Tang-Oxley Q. Qui W.R. Wang Y.-P. Mihindukulasuriya K.A. Afshar R. Tan T.-H. J. Biol. Chem. 1998; 273: 33561-33565Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Recent studies demonstrate that PP4 dephosphorylates RelA (NF-κB p65), primarily on Thr-435, and that this dephosphorylation is required for NF-κB activation induced by cisplatin (45Yeh P.Y. Yeh K.H. Chuang S.E. Song Y.C. Cheng A.L. J. Biol. Chem. 2004; 279: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). We have recently shown that PP4 takes part in relaying a TNF-α signal to the activation of the JNK pathway (46Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. VanHooser A. Hu M. C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and down-regulation of insulin receptor substrate 4 (47Mihindukulasuriya K.A. Zhou G. Qin J. Tan T.-H. J. Biol. Chem. 2004; (August 24, 10.1074/jbc.M408067200)PubMed Google Scholar). In our effort to investigate the molecular mechanism underlying the positive regulation of the JNK pathway by PP4, we found that PP4 interacted with and stabilized HPK1, leading to increased kinase activation of HPK1. In addition, PP4 enhanced the HPK1-induced JNK activation. We also observed that TCR stimulation enhanced the PP4-HPK1 interaction and that PP4 was involved in the kinase activation of HPK1 by TCR stimulation. These observations suggest that PP4 is a positive regulator for HPK1 and the HPK1-JNK signaling pathway. Reagents—[γ-32P]ATP, [32P]orthophosphate, and a mixture of [35S]methionine/cysteine were purchased from ICN Biomedicals (Irvine, CA). An ECL system was purchased from Amersham Biosciences. TNF-α was purchased from R&D Systems (Minneapolis, MN). Anti-HA antibody (12CA5) was purchased from Amersham Biosciences. Anti-FLAG (M2) and anti-β-actin were purchased from Sigma. Polyclonal anti-HPK1 (N-19), monoclonal anti-PP1, and anti-c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-JNK1 antibody (Ab 101) (48Chen Y.-R. Meyer C.F. Tan T.-H. J. Biol. Chem. 1996; 271: 631-634Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar), polyclonal anti-HPK1 antibody (Ab 484) (4Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar, 5Hu M. C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar), anti-CD3 antibody (28Han J. Kori R. Shui J.W. Chen Y.R. Yao Z. Tan T.-H. J. Biol. Chem. 2003; 278: 52195-52202Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and polyclonal anti-PP4 antibody (Ab 104) (46Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. VanHooser A. Hu M. C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) were previously described. All other chemical reagents were purchased from Sigma unless otherwise noted. Plasmids—The glutathione S-transferase-Jun-(1–79) was a gift from Dr. M. Karin (University of California, San Diego). pHA-JNK1 was a gift from Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada). pHA-ubiquitin was a gift from Dr. X.-H. Feng (Baylor College of Medicine, Houston, TX). pCMV-PP1 was a gift from Dr. A. H. Schonthal (University of Southern California) (49Jaramillo-Babb V.L. Sugarmans J.L. Scavetta R. Wang S.J. Berndt N. Born T.L. Glass C.K. Schonthal A.H. J. Biol. Chem. 1996; 271: 5988-5992Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). pMTSM-Myc-M3/6 was a gift from Dr. K. E. Davis (Oxford University) (50Theodosiou A.M. Rodrigues N.R. Nesbit M.A. Ambrose H.J. Paterson H. McLellan-Arnold E. Boyd Y. Leversha M.A. Owen N. Blake D.J. Ashworth A. Davies K.E. Hum. Mol. Genet. 1996; 5: 675-684Crossref PubMed Scopus (49) Google Scholar). FLAG-HPK1, FLAG-HPK1-M46, FLAG-HPK1-KD, FLAG-HPK1-CD, HA-HPK1-PR, and HA-HPK1-DR (8Ling P. Yao Z. Meyer C.F. Wang X.S. Oehl W. Feller S.M. Tan T.-H. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar), PP4, HA-PP4, and HA-PP4-RL (44Hu M. C.-T. Tang-Oxley Q. Qui W.R. Wang Y.-P. Mihindukulasuriya K.A. Afshar R. Tan T.-H. J. Biol. Chem. 1998; 273: 33561-33565Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and FLAG-GLK (51Diener K. Wang X.S. Chen C. Meyer C.F. Keesler G. Zukowski M. Tan T.-H. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (120) Google Scholar) were previously described. Cells and Transfection—Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 units/ml streptomycin/penicillin. Human embryonic kidney 293T (HEK293T) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin. HEK293T cells were plated at a density of either 1.5 × 105 cells/35-mm well or 1.5 × 106 cells/100-mm dish and transfected the next day using the modified calcium phosphate precipitation protocol (Specialty Media, Inc., Lavallette, NJ). Cells were transfected with plasmids encoding β-galactosidase (0.15 μg) in combination with an empty vector or various amounts of plasmids encoding phosphatases, phosphatase mutants, kinases, or kinase mutants as indicated in the figure legends to monitor the transfection efficiency. For those multiple transfections with identical component(s) (e.g. FLAG-HPK1 or FLAG-HPK1 plus PP4 in Fig. 6 and FLAG-HPK1 in Fig. 7A), we controlled transfection efficiency by performing FLAG-HPK1 transfection in 100-mm dishes and pooling the cells and aliquoting them into 6-well plates the next day after changing the medium. Jurkat cells (2 × 107/0.5 ml) were transiently transfected by electroporation (263 V, two pulses, and 10 ms) using a BTX Electro Square Porter T 820 (San Diego, CA). After electroporation, the cells were pooled (4 × 106/ml) and incubated overnight in RPMI 1640 supplemented with 5% fetal bovine serum. The cells were washed and resuspended in plain RPMI 1640 at a cell density of 1 × 108 cells/ml. 100 μl of the cell suspension (1 × 107) was aliquoted to tubes and incubated with 1 μl of anti-CD3 (OKT3) ascites for 10 min on ice. Goat anti-mouse antibody was added to cross-link the anti-CD3 (OKT3) ascites for 10 min with gentle rocking at 4 °C. Cell stimulations were performed at 37 °C for various time periods as indicated in the figure legend.Fig. 7HPK1 is subject to ubiquitin-targeted degradation. A, LLnL stabilizes HPK1. HEK293T cells (1.5 × 105 cells in 35-mm wells) were transfected with 0.5 μg of FLAG-HPK1. After 24 h, the cells were treated with various concentrations of LLnL as indicated for 16 h. The cell lysate was prepared and subjected to Western blot analysis with an anti-FLAG antibody (M2) (top). The blot was then reprobed with an anti-β-actin antibody (bottom). B, HPK1 is ubiquitinated. HEK293T cells (1.5 × 106 cells in 100-mm dishes) were transfected with vector alone, HA-ubiquitin (0.2 μg) alone, FLAG-HPK1 (5 μg) alone, or FLAG-HPK1 (5 μg) plus HA-ubiquitin (0.2 μg) with or without treatment of LLnL (20 μm) for 16 h. The cells were collected 40 h post-transfection. FLAG-HPK1 was immunoprecipitated (IP) with an anti-FLAG antibody (M2). The immunoprecipitates were then subjected to Western blotting (WB) using an anti-HA antibody (12CA5) (upper panel). The expression of FLAG-HPK1 was monitored by Western blot analysis with an anti-FLAG antibody (M2) (lower panel). C, PP4 inhibits HPK1 ubiquitination. HEK293T cells (1.5 × 106 cells in 100-mm dishes) were co-transfected with FLAG-HPK1 (5 μg) plus PP4 (10 μg) or PP4-RL (10 μg) in the presence of HA-ubiquitin (0.2 μg). The cells were collected 40 h post-transfection. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2). The immunoprecipitates were then subjected to Western blotting using an anti-HA antibody (12CA5) (upper panel). The expression levels of FLAG-HPK1, PP4, and PP4-RL were monitored by Western blot analysis with an anti-FLAG antibody (M2) and an anti-PP4 antibody (Ab 104), respectively (lower panels).View Large Image Figure ViewerDownload (PPT) Coimmunoprecipitation, Immunocomplex Kinase Assays, and Western Blot Analysis—Coimmunoprecipitation and immunocomplex kinase assays were performed as previously described (9Wang W. Zhou G. Hu M. C.-T. Yao Z. Tan T.-H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 10Zhou G. Lee S.C. Yao Z. Tan T.-H. J. Biol. Chem. 1999; 274: 13133-13138Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 52Chen Y.-R. Wang X. Templeton D. Davis R.J. Tan T.-H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). Western blot analysis was performed using an ECL kit according to the manufacturer's protocols (Amersham Biosciences). Labeling HPK1 in Vivo—HEK293T cells (1.5 × 106 cells in 100-mm dishes) were transfected with vector, FLAG-HPK1 (10 μg) alone, FLAG-HPK1 (10 μg) plus PP4 (10 μg), or FLAG-HPK1 (10 μg) plus PP4-RL (10 μg). Empty vector was used to normalize the amount of transfected DNA. 40 h post-transfection, the cells were maintained in the serum-free, phosphate-free DMEM for 2 h at 37 °C. The cells were then labeled in the phosphate-free DMEM supplemented with 10% dialyzed serum and 100 μCi of [32P]orthophosphate/ml for 4 h at 37 °C. The cells were washed with PBS twice to remove free [32P]orthophosphate. FLAG-HPK1 was immunoprecipitated with an anti-FLAG (M2) antibody and subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride and autoradiographed. The polyvinylidene difluoride membrane was then subjected to immunoblotting using an anti-FLAG (M2) antibody. Pulse-Chase Analysis—Pulse-chase experiments were performed using the [35S]methionine/cysteine mixture to monitor changes in the half-life of HPK1 in the presence or the absence of PP4. Briefly, 1.5 × 105 HEK293T cells/35-mm well were transfected with FLAG-HPK1 (0.2 μg) alone or with FLAG-HPK1 (0.2 μg) plus PP4 (2 μg). 36 h after transfection, the cells were starved in DMEM without methionine and cysteine for 1 h and then metabolically labeled with [35S]methionine/cysteine for 4 h. HEK293T cells were then chased in nonradioactive medium for the time periods as indicated. Cells were lysed, and the cell samples containing equal amounts of proteins were immunoprecipitated with an anti-FLAG antibody (M2). Immunocomplexes were collected with immobilized protein G-Sepharose beads and resolved on SDS-PAGE. The gels were dried and autoradiographed. HPK1 Interacts with PP4 through Its Proline-rich Region—We have previously found that PP4 acts as a positive regulator for the JNK pathway during TNF-α signaling and that PP4 probably exerts its effect on JNK in an indirect manner, since no direct PP4-JNK interaction was detected (46Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. VanHooser A. Hu M. C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The JNK pathway is composed of multiple kinases (53Whitmarsh A.J. Davis R.J. Trend Biochem. Sci. 1998; 23: 481-485Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 54Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1136) Google Scholar). In our effort to explore the molecular mechanism underlying the positive regulation of the JNK pathway by PP4, we sought to identify the target(s) of PP4 within the JNK pathway. We co-transfected PP4 into HEK293T cells with an individual upstream activating kinase of the JNK pathway, including MAPK kinases, MAPK kinase kinases, and MAPK kinase kinase kinases, followed by immunoprecipitation/Western blotting. We found that HPK1 is one of the upstream activating kinases that interacted with PP4. As shown in Fig. 1, FLAG-HPK1 was co-immunoprecipitated with PP4 when a specific anti-PP4 antibody (46Zhou G.

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