FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022379516> ?p ?o ?g. }

• W2022379516 endingPage "30996" @default.
• W2022379516 startingPage "30985" @default.
• W2022379516 abstract "The serine-threonine kinase PAK1 is activated by small GTPase-dependent and -independent mechanisms and promotes cell survival. However, the role of tyrosyl phosphorylation in the regulation of PAK1 function is poorly understood. In this study, we have shown that the prolactin-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo. Wild type, but not kinase-dead, JAK2 directly phosphorylates PAK1 in cells and in an in vitro kinase assay. PAK1 tyrosines 153, 201, and 285 were identified as sites of JAK2 tyrosyl phosphorylation by mass spectrometry and two-dimensional peptide mapping. Mutation of PAK1 tyrosines 153, 201, and 285 to phenylalanines individually or in combination implicated these PAK1 tyrosines in the regulation of PAK1 kinase activity. Tyrosyl phosphorylation by JAK2 significantly increases PAK1 kinase activity, whereas similar phosphorylation of the PAK1 Y153F,Y201F,Y285F mutant has no effect on PAK1 activity. Tyrosyl phosphorylation of wild type PAK1 decreases apoptosis induced by serum deprivation and staurosporine treatment and increases cell motility. In contrast, these parameters are unaltered in the PAK1 Y153F,Y201F,Y285F mutant. Our findings indicate that JAK2 phosphorylates PAK1 at these specific tyrosines and that this phosphorylation plays an important role in cell survival and motility. The serine-threonine kinase PAK1 is activated by small GTPase-dependent and -independent mechanisms and promotes cell survival. However, the role of tyrosyl phosphorylation in the regulation of PAK1 function is poorly understood. In this study, we have shown that the prolactin-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo. Wild type, but not kinase-dead, JAK2 directly phosphorylates PAK1 in cells and in an in vitro kinase assay. PAK1 tyrosines 153, 201, and 285 were identified as sites of JAK2 tyrosyl phosphorylation by mass spectrometry and two-dimensional peptide mapping. Mutation of PAK1 tyrosines 153, 201, and 285 to phenylalanines individually or in combination implicated these PAK1 tyrosines in the regulation of PAK1 kinase activity. Tyrosyl phosphorylation by JAK2 significantly increases PAK1 kinase activity, whereas similar phosphorylation of the PAK1 Y153F,Y201F,Y285F mutant has no effect on PAK1 activity. Tyrosyl phosphorylation of wild type PAK1 decreases apoptosis induced by serum deprivation and staurosporine treatment and increases cell motility. In contrast, these parameters are unaltered in the PAK1 Y153F,Y201F,Y285F mutant. Our findings indicate that JAK2 phosphorylates PAK1 at these specific tyrosines and that this phosphorylation plays an important role in cell survival and motility. PAK1 4The abbreviations used are: PAK1, p21-activated serine-threonine kinases; JAK2, Janus tyrosine kinase; PBD/CRIB, p21-binding domain/Cdc42/Rac interactive binding domain; SH2, Src homology 2; anti-Tyr(P), anti-phosphotyrosine antibody; STAT, signal transducer and activator of transcription; GST, glutathione S-transferase; HA, hemagglutinin; Ab, antibody; MS, mass spectrometry; siRNA, small interfering RNA; IP, immunoprecipitation.4The abbreviations used are: PAK1, p21-activated serine-threonine kinases; JAK2, Janus tyrosine kinase; PBD/CRIB, p21-binding domain/Cdc42/Rac interactive binding domain; SH2, Src homology 2; anti-Tyr(P), anti-phosphotyrosine antibody; STAT, signal transducer and activator of transcription; GST, glutathione S-transferase; HA, hemagglutinin; Ab, antibody; MS, mass spectrometry; siRNA, small interfering RNA; IP, immunoprecipitation. is a member of a conserved family of p21-activated serine-threonine kinases and is important for a variety of cellular functions, including cell morphogenesis, motility, survival, mitosis, and malignant transformation (for review see Refs. 1Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (879) Google Scholar, 2Zhao Z.S. Manser E. Biochem. J. 2005; 386: 201-214Crossref PubMed Scopus (221) Google Scholar, 3Kumar R. Gururaj A.E. Barnes C.J. Nat. Rev. Cancer. 2006; 6: 459-471Crossref PubMed Scopus (494) Google Scholar). The emerging roles of PAK1 in the regulation of multiple fundamental cellular processes have directed significant attention toward understanding how PAK1 activity is controlled. Autoinhibition of the PAK1 C-terminal catalytic domain by the N-terminal domain is a key mechanism of PAK1 regulation. Several layers of inhibition, involving dimerization and occupation of the catalytic cleft by contact between the N- and C-terminal domains, keep PAK1 kinase activity in check (4Lei M. Lu W. Meng W. Parrini M.C. Eck M.J. Mayer B.J. Harrison S.C. Cell. 2000; 102: 387-397Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Autoinhibition of PAK1 occurs in trans, meaning that the inhibitory domain of one PAK1 molecule interacts with the kinase domain of another PAK1 molecule (5Parrini M.C. Lei M. Harrison S.C. Mayer B.J. Mol. Cell. 2002; 9: 73-83Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Association of GTP-bound forms of Cdc42 and Rac1 with the PAK1 PBD/CRIB domain induces conformational changes in the N-terminal domain that no longer support its autoinhibitory function. In addition to Cdc42 and Rac1, PAK1 is activated by the binding of small GTPases, Rac2 and Rac3, as well as TC10, CHP, and Wrich-1 proteins (6Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1298) Google Scholar, 7Knaus U.G. Bokoch G.M. Int. J. Biochem. Cell Biol. 1998; 30: 857-862Crossref PubMed Scopus (159) Google Scholar, 8Mira J.P. Benard V. Groffen J. Sanders L.C. Knaus U.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 185-189Crossref PubMed Scopus (196) Google Scholar, 9Neudauer C.L. Joberty G. Tatsis N. Macara I.G. Curr. Biol. 1998; 8: 1151-1160Abstract Full Text Full Text PDF PubMed Google Scholar, 10Aronheim A. Broder Y.C. Cohen A. Fritsch A. Belisle B. Abo A. Curr. Biol. 1998; 8: 1125-1128Abstract Full Text Full Text PDF PubMed Google Scholar, 11Tao W. Pennica D. Xu L. Kalejta R.F. Levine A.J. Genes Dev. 2001; 15: 1796-1807Crossref PubMed Scopus (177) Google Scholar). PAK1 is a predominantly cytoplasmic protein, but is activated upon recruitment to the cell membrane. PAK1 membrane localization occurs through interaction with adaptor proteins Nck, Grb2, and PIX, all of which are activated by ligation of growth factor receptors (12Bokoch G.M. Wang Y. Bohl B.P. Sells M.A. Quilliam L.A. Knaus U.G. J. Biol. Chem. 1996; 271: 25746-25749Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 13Lu W. Katz S. Gupta R. Mayer B.J. Curr. Biol. 1997; 7: 85-94Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 14Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar, 15Zhao Z.S. Manser E. Loo T.H. Lim L. Mol. Cell Biol. 2000; 20: 6354-6363Crossref PubMed Scopus (315) Google Scholar). Membrane recruitment of PAK1 via adapter proteins and subsequent PAK1 activation may involve phosphorylation at Thr423 (a site that is also autophosphorylated when PAK1 is activated by Rac1 and Cdc42) by PDK1 (16King C.C. Gardiner E.M. Zenke F.T. Bohl B.P. Newton A.C. Hemmings B.A. Bokoch G.M. J. Biol. Chem. 2000; 275: 41201-41209Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) or interaction with lipids, such as sphingosine, that can activate PAK1 in a GTPase-independent manner (17Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1998; 273: 8137-8144Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In addition to PDK, several other protein kinases regulate PAK1. Thus, Akt1 phosphorylates PAK1 at Ser21, decreasing Nck binding to the PAK1 N terminus and stimulating PAK1 activity (18Tang Y. Zhou H. Chen A. Pittman R.N. Field J. J. Biol. Chem. 2000; 275: 9106-9109Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 19Zhao Z.S. Manser E. Lim L. Mol. Cell. Biol. 2000; 20: 3906-3917Crossref PubMed Scopus (127) Google Scholar). The p35-bound form of Cdk5, a neuron-specific protein kinase, associates with and phosphorylates PAK1 at Thr212 and inhibits PAK1 kinase activity (20Nikolic M. Chou M.M. Lu W. Mayer B.J. Tsai L.H. Nature. 1998; 395: 194-198Crossref PubMed Scopus (351) Google Scholar, 21Rashid T. Banerjee M. Nikolic M. J. Biol. Chem. 2001; 276: 49043-49052Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The cyclin B-bound form of Cdc2 also phosphorylates PAK1 at Thr212 (22Thiel D.A. Reeder M.K. Pfaff A. Coleman T.R. Sells M.A. Chernoff J. Curr. Biol. 2002; 12: 1227-1232Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Banerjee M. Worth D. Prowse D.M. Nikolic M. Curr. Biol. 2002; 12: 1233-1239Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) affecting PAK1 protein-protein interaction but not PAK1 activation (22Thiel D.A. Reeder M.K. Pfaff A. Coleman T.R. Sells M.A. Chernoff J. Curr. Biol. 2002; 12: 1227-1232Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The role of tyrosyl (Tyr) phosphorylation in the regulation of PAK1 function has not been well established. The non-receptor tyrosine kinase Etk/Bmx, a Tec family member, binds, phosphorylates, and activates PAK1 (24Bagheri-Yarmand R. Mandal M. Taludker A.H. Wang R.A. Vadlamudi R.K. Kung H.J. Kumar R. J. Biol. Chem. 2001; 276: 29403-29409Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In addition, a multiprotein complex containing Tyr-phosphorylated and highly active PAK was identified in constitutively activated v-ErbB receptor-transformed cells. Formation of this complex appeared to be Rho-dependent (25McManus M.J. Boerner J.L. Danielsen A.J. Wang Z. Matsumura F. Maihle N.J. J. Biol. Chem. 2000; 275: 35328-35334Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). A few studies also have reported Tyr phosphorylation of PAK2. PAK2 phosphorylation at Tyr130 by Src kinase strongly potentiates action of Cdc42/Rac1 on PAK2 (26Renkema G.H. Pulkkinen K. Saksela K. Mol. Cell. Biol. 2002; 22: 6719-6725Crossref PubMed Scopus (38) Google Scholar). Another tyrosine kinase, Abl, associates with PAK2 in vivo and decreases PAK2 kinase activity concomitantly with PAK2 Tyr phosphorylation on multiple sites (27Roig J. Tuazon P.T. Zipfel P.A. Pendergast A.M. Traugh J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14346-14351Crossref PubMed Scopus (42) Google Scholar). These studies suggest that the tyrosine kinase-dependent modulation of PAK1 activity may provide important mechanism(s) enabling cells to respond appropriately to different external stimuli. PAKs have been implicated in apoptosis, possessing either anti-apoptotic (PAK1, PAK4, and PAK5) or both pro-apoptotic and anti-apoptotic (PAK2) properties (for review see Refs. 1Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (879) Google Scholar and 3Kumar R. Gururaj A.E. Barnes C.J. Nat. Rev. Cancer. 2006; 6: 459-471Crossref PubMed Scopus (494) Google Scholar). PAK1 is activated by growth factors (epidermal growth factor and platelet-derived growth factor), and cytokines that promote cell survival. The survival signals induced by PAKs might be related to phosphorylation of the pro-apoptotic protein Bad (Bcl-2 family member), Raf-1, the forkhead transcriptional factor (FKHR), BimL (that interacts with and inactivates the anti-apoptotic protein Bcl-2), and dynein light chain 1 (18Tang Y. Zhou H. Chen A. Pittman R.N. Field J. J. Biol. Chem. 2000; 275: 9106-9109Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 28Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell. Biol. 2000; 20: 453-461Crossref PubMed Scopus (306) Google Scholar, 29Gnesutta N. Qu J. Minden A. J. Biol. Chem. 2001; 276: 14414-14419Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 30Cotteret S. Jaffer Z.M. Beeser A. Chernoff J. Mol. Cell. Biol. 2003; 23: 5526-5539Crossref PubMed Scopus (137) Google Scholar, 31Alavi A. Hood J.D. Frausto R. Stupack D.G. Cheresh D.A. Science. 2003; 301: 94-96Crossref PubMed Scopus (291) Google Scholar, 32Mazumdar A. Kumar R. FEBS Lett. 2003; 535: 6-10Crossref PubMed Scopus (88) Google Scholar, 33Vadlamudi R.K. Bagheri-Yarmand R. Yang Z. Balasenthil S. Nguyen D. Sahin A.A. den Hollander P. Kumar R. Cancer Cell. 2004; 5: 575-585Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The anti-apoptotic effects of PAK1 also may be mediated by activation of NFκB, a critical transcription factor involved in cell survival (34Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2445) Google Scholar). PAK1 mediates NFκB activation by Ras, Raf-1, and Rac1 and the expression of active PAK1 can stimulate NFκB on its own without activation of the inhibitor of κB kinases (35Frost J.A. Swantek J.L. Stippec S. Yin M.J. Gaynor R. Cobb M.H. J. Biol. Chem. 2000; 275: 19693-19699Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). PAK1 plays a key role in coordinating dynamic reorganizations of the actin and microtubule cytoskletons. PAK1 is localized in areas of the cortical actin cytoskeleton (36Dharmawardhane S. Sanders L.C. Martin S.S. Daniels R.H. Bokoch G.M. J. Cell Biol. 1997; 138: 1265-1278Crossref PubMed Scopus (196) Google Scholar); PAK1 kinase activity participates in directional motility (37Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 38Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (327) Google Scholar, 39Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (134) Google Scholar) and PAK1 directly phosphorylates cytoskeletal proteins, including LIM kinase (40Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (844) Google Scholar), p41-Arc (41Vadlamudi R.K. Li F. Barnes C.J. Bagheri-Yarmand R. Kumar R. EMBO Rep. 2004; 5: 154-160Crossref PubMed Scopus (106) Google Scholar), and filamin (42Vadlamudi R.K. Li F. Adam L. Nguyen D. Ohta Y. Stossel T.P. Kumar R. Nat. Cell Biol. 2002; 4: 681-690Crossref PubMed Scopus (262) Google Scholar). JAK2 is a tyrosine kinase that is activated by approximately two-thirds of the cytokine hematopoietin superfamily of receptors, including receptors for γ-interferons, most interleukins, ciliary neurotrophic factor, leptin, growth hormone, prolactin, leukemia inhibitory factor, oncostatin M, erythropoietin, and granulocyte macrophage-colony stimulating factor (43Murata T. Noguchi P.D. Puri R.K. J. Biol. Chem. 1995; 270: 30829-30836Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 44Argetsinger L.S. Carter-Su C. Horm. Res. 1996; 45: 22-24Crossref PubMed Scopus (36) Google Scholar, 45Roy B. Cathcart M.K. J. Biol. Chem. 1998; 273: 32023-32029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 46Parham C. Chirica M. Timans J. Vaisberg E. Travis M. Cheung J. Pflanz S. Zhang R. Singh K.P. Vega F. To W. Wagner J. O'Farrell A.M. McClanahan T. Zurawski S. Hannum C. Gorman D. Rennick D.M. Kastelein R.A. de Waal Malefyt R. Moore K.W. J. Immunol. 2002; 168: 5699-5708Crossref PubMed Scopus (1074) Google Scholar). The activation of JAK2 tyrosine kinase initiates a variety of downstream signaling events that lead to diverse physiological responses to cytokines, including regulation of body growth, hematopoiesis, satiety, lactation, and various components of immune function (47Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). The widely expressed SH2 domain-containing protein SH2-Bβ was initially identified as a binding partner and substrate of JAK2 (48Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar). SH2-Bβ binds preferentially via its SH2 domain to the tyrosyl-phosphorylated, active form of JAK2. SH2-Bβ binding dramatically increases JAK2 activity and enhances the tyrosyl phosphorylation of downstream targets of JAK2, such as STAT5 (49Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). Both JAK2 and PAK1 play critical roles in cell survival and their dysregulation can have negative consequences in terms of human disease. However, there is a gap between upstream JAK2 events and downstream PAK1 and PAK1-dependent cell survival. Elucidation of the mechanism by which signaling events downstream of JAK2 are regulated is therefore critical for our understanding of JAK2 signaling. Here we report that prolactin-activated JAK2 phosphorylates PAK1. We mapped three sites of JAK2-dependent phosphorylation of PAK1 and showed that phosphorylation at these sites significantly increases PAK1 kinase activity. In addition, these three phosphorylated tyrosines are required for maximal anti-apoptotic and motility-promoting PAK1 activities. Together, these results indicate that PAK1 is a new member of a JAK2-dependent signaling pathway playing an important role in cell survival and cell migration. Plasmids—cDNAs encoding myc-PAK1 T423E, myc-PAK1 K299R, HA-PAK1 WT, and GST-PAK1 were provided by Dr. Chernoff (Fox Chase Cancer Center, Philadelphia, PA); cDNAs encoding JAK2, JAK2 K882E, myc-SH2-Bβ, and myc-SH2-Bβ-(504-670) were provided by Dr. Carter-Su (University of Michigan, Ann Arbor, MI) (49Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). cDNA encoding mutant RacV12 was used with the permission of Dr. Hall (University College, London, United Kingdom). Individual tyrosines in PAK1 were mutated to phenylalanines using the QuikChange site-directed mutagenesis kit (Stratagene). The double mutant, PAK1 Y153F,Y201F was created by using PAK1 Y153F as a template and mutating Tyr201. The triple mutant PAK1 Y153F,Y201F,Y285F (PAK1 Y3F) was created by using PAK1 Y153F,Y201F as a template and mutating Tyr285. Mutations were confirmed by sequencing by the University of Michigan DNA Sequencing Core. PAK1 pSUPER-GFP targets the PAK1 mRNA and the mutated control PAK1 pSUPER-GFP were described early (50Li Z. Hannigan M. Mo Z. Liu B. Lu W. Wu Y. Smrcka A.V. Wu G. Li L. Liu M. Huang C.K. Wu D. Cell. 2003; 114: 215-227Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Antibody—Monoclonal anti-phosphotyrosine (anti-Tyr(P), clone 4G10) antibody (Ab) and polyclonal anti-JAK2 (used for Fig. 2A) and rabbit anti-mouse from Upstate Biotechnology, Inc. (Millipore), monoclonal anti-HA from Covance, polyclonal anti-PAK N-20 and monoclonal anti-Myc (9E10) from Santa Cruz Biotechnology, Inc., polyclonal anti-PAK1 from Cell Signaling, and monoclonal anti-actin (Sigma) were used for immunoprecipitation and immunoblotting. Monoclonal anti-HA Ab from Roche Applied Science was used for immunocytochemistry. Anti-JAK2 antiserum was provided by Dr. Carter-Su (51Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (822) Google Scholar) and used for immunoprecipitation, monoclonal anti-JAK2 Ab (number AHO1352, clone 691R5, BIOSOURCE) was used for immunoblotting. Anti-phospho-Thr423-PAK1 Ab was provided by Dr. Chernoff (39Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (134) Google Scholar), anti-SH2-B was provided by Dr. Carter-Su (48Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar), and anti-GIT1 was provided by Dr. Manser (15Zhao Z.S. Manser E. Loo T.H. Lim L. Mol. Cell Biol. 2000; 20: 6354-6363Crossref PubMed Scopus (315) Google Scholar). Cells—The stocks of prolactin-dependent Nb2 rat pre-T lymphoma cells (52Gout P.W. Cancer Res. 1987; 47: 1751-1755PubMed Google Scholar) were provided by Dr. Yu-Lee (Baylor College of Medicine) and Dr. Buckley (University of Cincinnati). The cells were grown in RPMI 1640 medium (Mediatech Cellgro) supplemented with 10% fetal bovine serum (HyClone), 10% horse serum (HyClone), 10-4 m 2-mercaptoethanol (Sigma), 1 mm glutamine (HyClone), 100 units of penicillin (HyClone) per ml, and 100 mg of streptomycin (HyClone) per ml. COS-7 and 293T cells were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle's medium (Mediatech Cellgro) supplemented with 10% fetal calf serum (for COS-7) or 10% calf serum (Mediatech Cellgro, for 293T cells), 1 mm glutamine, 100 units of penicillin/ml, and 100 mg of streptomycin/ml. Immortalized normal mammary epithelial (HME) cells were provided by Drs. Ethier (Karmanos Cancer Institute, MI) and Band (Northwestern University, IL). The cells were grown in Ham's F-12 medium (Mediatech Cellgro) supplemented with 5 μg/ml insulin (Sigma), 1 μg/ml hydrocortisone (Sigma), 10 ng/ml epidermal growth factor (Sigma), 100 ng/ml cholera toxin (Sigma), 2.5 μg/ml plasmocin (Amaxa), 2.5 μg/ml fungizone (Invitrogen), 5 μg/ml gentamicin (Invitrogen), and 5% fetal bovine serum. Co-immunoprecipitation and Immunoblotting—For prolactin treatment, the Nb2 cells were rendered quiescent by growth in maintenance medium with 3% horse serum and no fetal bovine serum in the presence or absence of 50 μm AG 490 (Calbiochem) for 24 h at 1 × 107 cells/ml and treated with 10 nm ovine prolactin for 10 min (purchased from Dr. Parlov, National Hormone and Peptide Program, NIDDK, National Institutes of Health). PAK1 was immunoprecipitated with anti-PAK1 Ab and protein A-agarose, resolved by SDS-PAGE followed by immunoblotting with anti-Tyr(P), anti-PAK1, and anti-JAK2 Abs. 293T cells were transiently transfected using calcium phosphate precipitation. HA-PAK1 was overexpressed with JAK2 or JAK2 K882E, and SH2-Bβ. The cells were serum deprived overnight and PAK1 was immunoprecipitated with anti-HA, rabbit anti-mouse IgG, and protein A-agarose and resolved by SDS-PAGE followed by immunoblotting with the indicated AB. In Vitro Kinase Assay—HA-PAK1 was overexpressed with JAK2 or JAK2 K882E, and SH2-Bβ in 293T cells. After overnight deprivation, the cells were lysed, HA-PAK1 was immunoprecipitated with anti-HA and immune complexes were collected using protein A-agarose. The immobilized PAK1 was incubated at 30 °C for 30 min in 50 μl of kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol) containing 5 μCi of [γ-32P]ATP (ICN), 10 μg/ml aprotinin, and 10 μg/ml leupeptin (39Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (134) Google Scholar). Precipitates were washed extensively with lysis buffer and protein A-agarose-PAK1-JAK2 complexes were pelleted. Radiolabeled proteins were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography followed by immunoblotting with anti-Tyr(P), anti-JAK2, and anti-PAK1 Abs. For pull-down assays, JAK2 or JAK2 K882E were overexpressed with SH2-Bβ-(504-670) in 293T cells, and immunoprecipitated with anti-JAK2. The immobilized JAK2 was incubated with 1 μg of GST-PAK1 in the kinase buffer as described above and PAK1 phosphorylation was assessed. To assess PAK1 in vitro kinase activity, HA-tagged wild type or mutated PAK1 or Myc-tagged PAK1 K299R were immunoprecipitated with anti-HA or anti-Myc Abs from 293T cells and subjected to in vitro kinase assay in the presence of 1 μCi of [γ-32P]ATP, 1 μm ATP, and 5 μg of histone H4 (substrate of PAK1). Relative levels of incorporation of 32P into histone H4, an indicator of phosphorylation, were assessed by autoradiography and estimated by phosphorimager. The same membrane was blotted with anti-HA or anti-Myc to assess the amount of PAK1 for each condition. Nitrocellulose patterns were scanned and the amount of PAK1 was quantified using Multi-Analyst (Bio-Rad) software. Relative PAK1 kinase activity was then normalized by the amount of immunoprecipitated PAK1 for each lane. Phosphopeptide Mapping and Phosphoamino Acid Analysis—Wild type and mutant forms of HA-tagged PAK1 were co-expressed with JAK2 and SH2-Bβ in 293T cells. The cells were deprived of serum overnight. PAK1 were immunoprecipitated with anti-HA and incubated with 0.5 mCi of [γ-32P]ATP as described above. Proteins will be resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. The region of the nitrocellulose containing 32P-labeled PAK1 was excised, soaked in 500 μl of 0.5% polyvinylpyrrolidone in 100 mm acetic acid at 37 °C for 30 min, and digested with 5 μg of methylated trypsin (Promega) for 4 h at 37 °C. Digested peptides were lyophilized, oxidized in performic acid, and re-lyophilized. Peptides were then separated first by thin layer electrophoresis and then in the second dimension by ascending chromatography (53Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar, 54O'Brien K.B. Argetsinger L.S. Diakonova M. Carter-Su C. J. Biol. Chem. 2003; 278: 11970-11978Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). 32P labeling peptides will be visualized by autoradiography. Detection of Phosphorylation Sites by Tandem MS—HA- or Myc-tagged PAK1 was overexpressed in 293T cells with JAK2 and SH2-Bβ. The cells were serum deprived overnight and PAK1 was immunoprecipitated with anti-HA or anti-Myc and resolved by SDS-PAGE. Gel slices containing PAK1 were digested with 5 ng/μl sequencing grade modified trypsin (Promega) in 25 mm ammonium bicarbonate containing 0.01% n-octylglucoside for 18 h at 37 °C. Peptides were eluted from the gel slices with 80% acetonitrile, 1% formic acid. Tryptic digests were separated by capillary high pressure liquid chromatography (C18, 75 μm inner diameter Picofrit column, New Objective) using a flow rate of 100 nl/min over a 3-h reverse phase gradient and analyzed using a LTQ linear Ion Trap LC/MSn system (Thermo Electron). Resultant MS/MS spectra were matched against the PAK1 sequence using TurboSequest (BioWorks 3.2, Thermo Electron) with fragment ion tolerance <0.3 and amino acid modification variables including phosphorylation (80 Da) of Ser, Thr, and Tyr, and oxidation (16 Da) of Met. PAK1 Gene Silencing—For synthesis of PAK1 siRNA in vivo, COS-7 cells were transiently transfected with cDNA encoding PAK1 pSUPER-GFP targeting the PAK1 mRNA or control pSUPER-GFP that produces a siRNA that is 2 base pairs different from the PAK1 siRNA using Nucleofector kit V (Amaxa Biosystmes) according to the manufacturer's protocol. After 72 h, cells were analyzed for PAK1 by immunoprecipitation and immunoblotting with anti-PAK1. Apoptosis Assay—COS-7 cells grown on coverslips were transfected with cDNA encoding myc-PAK1 T423E, myc-PAK1 K299R, HA-PAK1 WT, HA-PAK1 Y3F, JAK2, or JAK2 K882E as indicated using FuGENE 6 (Roche) according to the manufacturer's protocol. In 24 h the cells were serum-deprived and treated with 50 nm staurosporine. In 16 h the cells were fixed with 4% formaldehyde solution for 30 min, permeabilized with 0.1% Triton, and incubated with 2% human serum for 15 min for blocking of nonspecific staining. Next, the coverslips were incubated with anti-HA or anti-Myc Ab followed by rabbit anti-mouse/Oregon Green AB (Molecular Probes, Inc.) and 4′,6-diamidino-2-phenylindole (Molecular Probes, Inc.). After washing, the coverslips were incubated with the TUNEL reaction mixture (Roche) according to the manufacturer's protocol. The images presented are representative of at least 3 separate experiments. In all cases staining by secondary antibody reagent alone was negligible (not shown). Apoptotic index was calculated as a ratio of amount of TUNEL- and tag-positive cells to the amount of tag-positive cells (for at least 100 cells for each experiment). Each experiment was repeated at least three times, data were pooled, the average apoptotic index for each condition was plotted and analyzed using two-tailed unpaired t test. When individual experiments were analyzed, the results were indistinguishable from those obtained from the pooled data. Differences were considered to be statistically significant at p < 0.05. Results are expressed as the mean ± S.E. Phagokinetc Assay—For the phagokinetic assay, HME cells were transfected with cDNA encoding myc-PAK1 T423E, HA-PAK1 WT, HA-PAK1 Y3F, JAK2, or JAK2 K882E as indicated using Express-Fect (Denville Scientific, Inc.) and plated on colloid gold-covered coverslips 48 h after transfection (55Albrecht-Buehler G. Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar, 56Diakonova M. Gunter D.R. Herrington J. Carter-Su C. J. Biol. Chem. 2002; 277: 10669-10677Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Cells remove particles while they move, thereby producing areas that are free of colloid gold. The incubation time (16 h) was experimentally determined to avoid the overlapping of the particle-free areas produced by neighbor cells. In 16 h the coverslips were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 15 min, and incubated with anti-HA or anti-Myc followed by goat anti-mouse fluorescein isothiocyanate. F-actin was stained with Texas Red-phalloidin. Individual transfected cells were located with a fluorescein isothiocyanate filer set using a Zeiss Axiovert 200 microscope. Differential interference contrast images were collected and particle-free areas were quantified using Image Tool software. The particle-free area was measured in three independent experiments for about 50 cells. The phagokinetic index was cal" @default.
• W2022379516 created "2016-06-24" @default.
• W2022379516 creator A5003283869 @default.
• W2022379516 creator A5026005041 @default.
• W2022379516 creator A5062118117 @default.
• W2022379516 creator A5065846931 @default.
• W2022379516 creator A5077963098 @default.
• W2022379516 date "2007-10-01" @default.
• W2022379516 modified "2023-10-14" @default.
• W2022379516 title "JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions" @default.
• W2022379516 cites W1490646520 @default.
• W2022379516 cites W1528493806 @default.
• W2022379516 cites W1555510730 @default.
• W2022379516 cites W1576582350 @default.
• W2022379516 cites W1637051517 @default.
• W2022379516 cites W1826874877 @default.
• W2022379516 cites W1847326477 @default.
• W2022379516 cites W1932148910 @default.
• W2022379516 cites W1964843584 @default.
• W2022379516 cites W1964957919 @default.
• W2022379516 cites W1969814333 @default.
• W2022379516 cites W1971298668 @default.
• W2022379516 cites W1974656027 @default.
• W2022379516 cites W1974937739 @default.
• W2022379516 cites W1987821958 @default.
• W2022379516 cites W1988683816 @default.
• W2022379516 cites W1989290158 @default.
• W2022379516 cites W1989987282 @default.
• W2022379516 cites W2002782759 @default.
• W2022379516 cites W2003597023 @default.
• W2022379516 cites W2004375684 @default.
• W2022379516 cites W2006668021 @default.
• W2022379516 cites W2009461629 @default.
• W2022379516 cites W2011756230 @default.
• W2022379516 cites W2022807358 @default.
• W2022379516 cites W2022827874 @default.
• W2022379516 cites W2022850508 @default.
• W2022379516 cites W2024952351 @default.
• W2022379516 cites W2034180982 @default.
• W2022379516 cites W2037040435 @default.
• W2022379516 cites W2040567131 @default.
• W2022379516 cites W2048702755 @default.
• W2022379516 cites W2048778098 @default.
• W2022379516 cites W2055289589 @default.
• W2022379516 cites W2058381459 @default.
• W2022379516 cites W2067677546 @default.
• W2022379516 cites W2067854585 @default.
• W2022379516 cites W2067894718 @default.
• W2022379516 cites W2069078564 @default.
• W2022379516 cites W2069556423 @default.
• W2022379516 cites W2070190928 @default.
• W2022379516 cites W2070512478 @default.
• W2022379516 cites W2070629020 @default.
• W2022379516 cites W2075725057 @default.
• W2022379516 cites W2080145523 @default.
• W2022379516 cites W2081414779 @default.
• W2022379516 cites W2084606144 @default.
• W2022379516 cites W2086405676 @default.
• W2022379516 cites W2091931544 @default.
• W2022379516 cites W2102185138 @default.
• W2022379516 cites W2106140806 @default.
• W2022379516 cites W2108381748 @default.
• W2022379516 cites W2112111997 @default.
• W2022379516 cites W2113674401 @default.
• W2022379516 cites W2114207292 @default.
• W2022379516 cites W2119290720 @default.
• W2022379516 cites W2121810961 @default.
• W2022379516 cites W2122153913 @default.
• W2022379516 cites W2127048642 @default.
• W2022379516 cites W2127441616 @default.
• W2022379516 cites W2129191533 @default.
• W2022379516 cites W2129277563 @default.
• W2022379516 cites W2131129488 @default.
• W2022379516 cites W2131206883 @default.
• W2022379516 cites W2132306888 @default.
• W2022379516 cites W2132948080 @default.
• W2022379516 cites W2133508703 @default.
• W2022379516 cites W2150647809 @default.
• W2022379516 cites W2156566018 @default.
• W2022379516 cites W2160584487 @default.
• W2022379516 cites W2161340336 @default.
• W2022379516 cites W2165183041 @default.
• W2022379516 cites W2167630501 @default.
• W2022379516 cites W2470223656 @default.
• W2022379516 doi "https://doi.org/10.1074/jbc.m701794200" @default.
• W2022379516 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17726028" @default.
• W2022379516 hasPublicationYear "2007" @default.
• W2022379516 type Work @default.
• W2022379516 sameAs 2022379516 @default.
• W2022379516 citedByCount "44" @default.
• W2022379516 countsByYear W20223795162012 @default.
• W2022379516 countsByYear W20223795162013 @default.
• W2022379516 countsByYear W20223795162014 @default.
• W2022379516 countsByYear W20223795162015 @default.
• W2022379516 countsByYear W20223795162016 @default.
• W2022379516 countsByYear W20223795162017 @default.
• W2022379516 countsByYear W20223795162019 @default.
• W2022379516 countsByYear W20223795162020 @default.

• next