SH2/SH3 Adaptor Proteins Can Link Tyrosine Kinases to a Ste20-related Protein Kinase, HPK1

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2·HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases. Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2·HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases. In the budding yeast, Saccharomyces cerevisiae, the Ste20 serine/threonine protein kinase regulates a mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GST, glutathioneS-transferase; HA, hemagglutinin; HPK1, hematopoietic progenitor kinase 1; Tyr(P), phosphotyrosine; SAPK/JNK, stress-activated protein kinase/Jun N-terminal kinase; SH3 domain, Src homology 3 domain; R, receptor; SPR, surface plasmon resonance; DTT, dithiothreitol; PDGF, platelet-derived growth factor; EGF, epidermal growth factor. pathway, composed of the Ste11 (MEK kinase), Ste7 (MEK), and Fus3/Kss1 (MAPK) protein kinases (1Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (867) Google Scholar). Ste20 controls the mating response of haploid cells and is positioned by genetic epistasis analysis between heterotrimeric G proteins activated by the α factor mating pheromone and the MAPK cassette (2Zhao Z.S. Leung T. Manser E. Lim L. Mol. Cell. Biol. 1995; 15: 5246-5257Crossref PubMed Scopus (161) Google Scholar). In addition to its role in the mating pathway, yeast Ste20 participates in the development of nitrogen-deprived diploid cells into a filamentous pseudohyphal form and in the ability of haploid cells to invade an agar substrate (3Peter M. Neiman A.M. Park H.-O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (193) Google Scholar, 4Roberts R.L. Fink G.R. Gene Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (529) Google Scholar). Ste20 specifically associates through an N-terminal regulatory sequence with the small GTPase Cdc42 (2Zhao Z.S. Leung T. Manser E. Lim L. Mol. Cell. Biol. 1995; 15: 5246-5257Crossref PubMed Scopus (161) Google Scholar), an interaction that results in the localization of cytosolic Ste20 to specific membrane sites. Of interest, the interaction of Ste20 with Cdc42 is essential for cell invasion and pseudohyphal formation, as well as for an effect of Ste20 in stabilizing septin-containing microfilaments during cytokinesis, but is dispensable for the mating response (3Peter M. Neiman A.M. Park H.-O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (193) Google Scholar). Thus Ste20 appears to have pleiotropic functions in the yeast cell and to have both multiple upstream activators and downstream biological outputs. Recent work has identified a number of mammalian kinases whose catalytic domains are closely related in sequence to that of yeast Ste20 (5Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). Among these, members of the PAK family share the same overall structure as Ste20, consisting of a C-terminal kinase domain, and an N-terminal regulatory region that contains binding motifs for the small GTPases Cdc42 and Rac1 (6Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar), as well as for the second of the three SH3 domains of the adaptor protein Nck. Interactions with these signaling proteins may both relocalize PAK1 and PAK3 to the membrane and stimulate their kinase activity (7Lu W. Katz S. Gupta R. Mayer B.J. Curr. Biol. 1997; 7: 85-94Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 8Galisteo M.L. Chernoff J. Su Y.C. Skolnik E.Y. Schlessinger J. J. Biol. Chem. 1996; 271: 20997-21000Abstract Full Text PDF PubMed Scopus (237) Google Scholar). PAK kinases have been implicated in the regulation of a MAPK cassette that responds to stressful stimuli and results in activation of the stress-activated protein kinases (SAPK/JNK) and p38 (5Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar, 6Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 9Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). PAK1 may also be involved in the control of actin stress fiber disassembly and in reorganization of focal complexes (10Manser 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, 11Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenstrom P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). A distinct family of Ste20-related mammalian protein kinases including GC kinase, HPK1, KHS, NIK, and MST1 and -2 consists of an N-terminally located kinase domain and a C-terminal tail, which is proposed to have a regulatory function (6Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 12Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. 1995; PubMed Scopus Google Scholar, T.-H. Gene Dev. 1996; PubMed Scopus Google Scholar, M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar, Chernoff J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. Skolnik E.Y. EMBO J. 1997; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar). PAK kinases, GC kinase, HPK1, NIK, and have the to the pathway C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. 1995; PubMed Scopus Google Scholar, T.-H. Gene Dev. 1996; PubMed Scopus Google Scholar, M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google J. 1997; PubMed Scopus Google Scholar), may well have biological The as well as the related kinases of the family are of interest, as are activated in response to including and and (5Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). the of and p38 activation are not well have been to growth and the of with a role for stress pathways in cellular in cell A.M. J. A. M. Zon L.I. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. B. S. E. Zon L.I. Kyriakis J.M. A. 1996; PubMed Scopus Google Scholar, M. J. Davis R.J. 1995; 270: Scopus Google Scholar, E. E. Zon L. Kyriakis J. Woodgett J.R. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). factor to be of factor activation M. Cell. 1996; 87: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Woodgett J.R. M. 1997; Scopus Google Scholar). HPK1 is a serine/threonine protein kinase that is during development and to hematopoietic in the T.-H. Gene Dev. 1996; PubMed Scopus Google Scholar, M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar). HPK1 with a downstream kinase, which has an N-terminal SH3 in can stimulate leading to activation of M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar, A. P. S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google N. Woodgett N. EMBO J. 1996; 15: PubMed Scopus Google Scholar). Here we have investigated through which cell surface might be to HPK1. HPK1 contains four proline-rich motifs in its C-terminal tail, that might associate with cytoplasmic signaling proteins that SH3 Here we that HPK1 SH2/SH3 adaptor In a Grb2 bound through its SH3 domains to HPK1. EGF stimulation the Grb2·HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking and HPK1 These and related suggest that SH2/SH3 adaptors such as Grb2 can tyrosine kinases to HPK1, a Ste20-related serine/threonine protein kinase. which tyrosine kinase to the may provide a between biochemical pathways and a mechanism for the proline-rich region of HPK1 as in into T. Chen Gene Dev. 7: PubMed Scopus Google Scholar). SH3 domains into the as M. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The proteins of Grb2 and and SH3 domain I. R. P. J. Cell. Full Text PDF PubMed Scopus Google Scholar). HPK1 and kinase M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar). of the and of into a the Grb2 with specific M. Gupta R. Mayer B. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google a J. I. T. Mol. Cell. Biol. PubMed Scopus Google Scholar). between the binding domain of and the SH3 and the activation domain of to of the HPK1 proline-rich into S. and by the in that during of and by mating and yeast the activity the yeast by an with activation as by the of the the and through a in and a composed of by and The with by and The of the by analysis and plasmon analysis to binding to Grb2 in a a A protein of HPK1 in the surface of a surface following for of C.M. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of Grb2 of in 3 and DTT, and a of the surface the and the The of Grb2 bound the surface plasmon a during the and the bound to of Grb2 the surface and N-terminal the to the of HPK1. did not with complex and used for in associates with the activated EGF Cos1 cells transfected with Grb2 with HPK1. stimulated with EGF as Cell with and with to the complex cell with to with to autophosphorylated EGF receptor EGF stimulation did not affect of the Cos1 cells with and HPK1 and stimulated with EGF Cell with to the EGF receptor HPK1 and the with to the EGF EGF stimulation induced the of both and HPK1 with the EGF Cos1 cells with and HPK1 and stimulated with EGF Cell with receptor and with to HPK1. cell also by for HPK1 the of HPK1 with the activated EGF Cos1 cells transfected with HPK1 and in and stimulated with EGF as Cell with and with receptor The of the of HPK1 with the EGF receptor and Shc cell with and with to cell with to the of HPK1 C-terminal the to of HPK1. used for HPK1 analysis is tyrosine in cells. Cos1 cells transfected with in with HPK1. The cells in the of for by EGF stimulation as HPK1 with to the HPK1 proline-rich phosphorylation of HPK1 by the with The same and with to the of HPK1 in to the proline-rich region of HPK1 a protein HPK1 The of I. J. T. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). induced with The cell by in DTT, The to for The with and the with the in and used for in vitro binding Cell cells with of protein to in and The three and for with by and to a HPK1 by analysis with Cos1 cells transfected with of the in with an HPK1 of growth in with the cells in for to EGF the cells three in and with A and HPK1 by and the complexes to analysis as Cos1 cells transfected with HPK1 in with for tyrosine kinases, as The cells in to EGF and HPK1 specific to the proline-rich region of HPK1. phosphorylation of HPK1 by the protein with with A of four proline-rich are located in the C-terminal tail of HPK1, in the region following the kinase domain of these proline-rich and sequence homology with motifs located in the tail of that Grb2 SH3 domains M. R. J. T. PubMed Scopus Google Scholar, N. A. R. Skolnik E. P. B. Schlessinger J. PubMed Scopus Google Scholar, Chem. Biol. 1995; Full Text PDF PubMed Scopus Google Scholar). These HPK1 motifs to the sequence for SH3 binding in the S. Chen 1994; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). analysis has that such a in which of the SH3 domain, the a The SH3 domains of adaptors such as Grb2 a the as in the HPK1 proline-rich motifs. used the yeast to the ability of the proline-rich of HPK1 to specific SH3 A of HPK1 four proline-rich bound to both N-terminal and C-terminal SH3 domains of to a the three SH3 to the SH3 domain of the kinase, and to the SH3 A analysis of these interactions HPK1 with three of the proline-rich motifs in binding the Grb2 domain with a the the Grb2 domain did The Grb2 domain an interaction with both Grb2 SH3 domains bound a and The ability of the Grb2 SH3 domains to HPK1 with distinct that both SH3 domains of Grb2 might HPK1. in the yeast the three SH3 domains with of the HPK1 proline-rich that might also multiple HPK1 motifs. have M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google that HPK1 which in can and stimulate The SH3 domain with HPK1 the and sites. these interactions proteins specific SH3 domains with cell cells. The proteins and and for the of HPK1 analysis The Grb2 SH3 domains a complex with HPK1, in the N-terminal SH3 domain A protein Grb2 bound HPK1 SH3 domain that the SH3 domains of Grb2 may HPK1 in a as by the A the three SH3 domains also bound HPK1, proteins the SH3 bound of HPK1 in that vitro interactions of HPK1 with the Grb2 and SH3 domains are the of HPK1 that might interact with we a of to the proline-rich of HPK1 and their ability to Grb2 binding to the tail of HPK1 as surface plasmon In a protein to a surface and the following of Grb2 in the and of in 3 the that region may be a for Grb2 binding HPK1. and also for of and did not Grb2 binding to HPK1. In we have that a proline-rich tail the interaction to a as the HPK1 not These are with results for the binding of Grb2 to proline-rich M. R. J. T. PubMed Scopus Google Scholar, Chem. Biol. 1995; Full Text PDF PubMed Scopus Google and suggest that Grb2 may in a to both HPK1 and the of an complex in vitro and identified specific proline-rich motifs in HPK1 that can interaction through of Grb2 SH3 we investigated such a complex can form in an of Grb2 in Cos1 cells in with HPK1. of HPK1 with we for the of Grb2 by analysis with in the of HPK1. of both HPK1 and in the of a that HPK1 and Grb2 can associate in mammalian cells. The role of the Grb2 SH3 domains in binding to HPK1 in Cos1 cells SH3 in which an essential for SH3 domain binding with in the N-terminal C-terminal SH3 in both SH3 domains M. Gupta R. Mayer B. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). of Grb2 SH3 domain in a in the and of both SH3 domains complex These results suggest that the SH3 domains of Grb2 synergistically to HPK1 in mammalian cells. the signals HPK1 activity are transfected into Cos1 cells HPK1, Ste20-related kinases, is as a of the pathway T.-H. Gene Dev. 1996; PubMed Scopus Google Scholar, M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar). has the analysis of signals that might HPK1. the interaction of HPK1 with the SH3 domains of Grb2 and that HPK1 might be by such as activated growth factor the Shc that the Grb2 domain through motifs J. P. T. M. T. R. S. R.A. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, T. 1994; PubMed Scopus Google Scholar). we have used the EGF receptor as a to the possible of receptor tyrosine kinase activation HPK1. EGF stimulation of a complex Grb2 and is to the autophosphorylated EGF receptor M. R. J. T. PubMed Scopus Google N. A. R. Skolnik E. P. B. Schlessinger J. PubMed Scopus Google through of the activated receptor by the Grb2 HPK1 contains C-terminal proline-rich motifs closely related to in the tail of and to we EGF stimulation binding of the complex to the autophosphorylated EGF EGF stimulation of Cos1 cells HPK1 and Grb2 did not affect the binding of HPK1 to Grb2 following stimulation of these cells with EGF, both and HPK1 with the EGF receptor as by of both HPK1 and with the activated receptor between HPK1 and the EGF receptor also of the EGF receptor and analysis with The interaction not HPK1 kinase a HPK1 M. A. N. T. Woodgett J.R. EMBO J. 1996; 15: PubMed Scopus Google with the EGF receptor well as HPK1 Grb2 might the of HPK1 with the EGF receptor, we the effect of of Grb2 of the receptor The of the EGF receptor that with HPK1 in Cos1 cells transfected with and as with cells Grb2 that Grb2 can the interaction between the EGF receptor and HPK1. In addition to a direct interaction with the EGF receptor, Grb2 through its domain to proteins in the Shc adaptor P. S. T. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. I. T. 1994; Google Scholar). with the that Grb2 might interaction between proteins and HPK1, EGF stimulation induced the of a complex Shc proteins and HPK1, which by of Grb2 These that EGF the of a the yeast analysis as well as in vitro binding with proteins 3 that adaptor might also have the to interact with HPK1 in of Cos1 cells that been transfected with HPK1 with Nck. to with HPK1 transfected Cos1 cells. the Grb2·HPK1 the interaction between and HPK1 stimulated by In of Grb2 the interaction between and HPK1, that these SH2/SH3 adaptors can in for HPK1 The recruitment of the complex to the activated EGF receptor might potentially in phosphorylation of HPK1 HPK1 Cos1 cells that been transfected with HPK1 and and stimulated with phosphorylation of HPK1 by with A protein in to HPK1 specifically in cells that been transfected with HPK1. in transfected cells in cells that not been transfected with HPK1 and stimulated with The the cells with the tyrosine to EGF These results suggest that HPK1 can be in cells and that is by a tyrosine activity in tyrosine phosphorylation of HPK1 be mediated by growth factor HPK1 with the receptor for platelet-derived growth factor stimulation of these cells in HPK1 tyrosine phosphorylation In we that HPK1 in cells that been with with activated of cytoplasmic tyrosine kinases J. A. T. S. A. PubMed Scopus Google whose are in hematopoietic cells S. A. EMBO J. PubMed Scopus Google Scholar, T. 1995; PubMed Scopus Google and that HPK1 is potentially by a of tyrosine kinases. have investigated possible through which the Ste20-related protein kinase HPK1 might be to cell surface results have that proline-rich motifs in the tail of HPK1 can in to the SH3 domains of the adaptor proteins Grb2 and Nck. a we have that Grb2 can HPK1 to the activated EGF These a which to the regulation of HPK1 in hematopoietic cells and suggest that adaptor proteins such as Grb2 may distinct biochemical signaling pathways protein serine/threonine kinases. SH2/SH3 adaptor proteins such as and activated growth factor and cytoplasmic docking proteins to downstream with proline-rich motifs. A of genetic and biochemical has that an important biological function of mammalian Grb2 and its is to tyrosine kinases to M. R. J. T. PubMed Scopus Google Scholar, N. A. R. Skolnik E. P. B. Schlessinger J. PubMed Scopus Google Scholar, Chem. Biol. 1995; Full Text PDF PubMed Scopus Google and to the GTPase and a downstream MAPK cassette composed of the and kinase protein kinases R. Cell. 1995; 80: Full Text PDF PubMed Scopus Google and T. 1995; PubMed Scopus Google Scholar). the function of is to the pathway, biochemical that the Grb2 SH3 domains have binding and functions and that multiple of Grb2 may in mammalian cells. interactions of the Grb2 domain with the GTPase may receptor I. R. P. J. Cell. Full Text PDF PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). In addition to and the Grb2 SH3 domains have been to proteins with proline-rich L. van J. 1996; Google Scholar, R. Gene Dev. 1994; 8: PubMed Scopus Google Scholar, A. T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. T. N. E. R. Woodgett J. Avruch J. Kyriakis J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). these Grb2 binding have multiple SH3 binding motifs related to in these results suggest that adaptors such as Grb2 may the of multiple signaling complexes in a and a of signaling downstream of tyrosine kinases. Here we that HPK1, a Ste20-related kinase, to Grb2 SH3 both in vitro and in The of HPK1 is to that of with a N-terminal catalytic domain and an extended C-terminal region multiple proline-rich motifs three of the proline-rich motifs in HPK1 are to Grb2 in with the that these HPK1 proline-rich motifs might in these HPK1 bound Grb2 in vitro 3 of the Grb2 SH3 as with HPK1 mammalian cell 3 Grb2 SH3 domains with specific proline-rich HPK1 in the yeast with distinct Grb2 bound to HPK1 in Cos1 cells. of Grb2 with HPK1 that both Grb2 SH3 domains be with the that the SH3 domains may distinct and synergistically to HPK1. HPK1 proteins in its interactions with the Grb2 domain, which to proline-rich and is dispensable for signaling to the pathway Chem. Biol. 1995; Full Text PDF PubMed Scopus Google Scholar, T. B. T. E. EMBO J. 1995; PubMed Scopus Google Scholar). may potentially be by the the in of the HPK1 and proline-rich motifs. The Grb2 domain to with as in of the proline-rich motifs B. S. J. A. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and the and of HPK1 that associate with have The Grb2 domain, in bound the in HPK1, which has a by a and and These results are with the that the domain to the in HPK1, the domain to the HPK1 appears to to Grb2 in can also associate with SH3 domains both in vitro and in 3 and The that binding by of Grb2 that these adaptors may for binding to the same HPK1, as by the yeast analysis The HPK1 complexes in the of specific adaptors and the of their SH3 domains for HPK1 motifs. A between Grb2 and binding to HPK1 is that the interaction of HPK1 with Grb2 appears its with is stimulated by EGF stimulation A and A of binding has been for the of Grb2 with in activated cells Y.C. J. 1995; PubMed Scopus Google Scholar). The for the interaction between and HPK1 is but might tyrosine phosphorylation of of HPK1. we have that tyrosine phosphorylation of the SH2/SH3 adaptor can interactions M. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The that Grb2 through its SH3 domains to HPK1 to Grb2 as a to tyrosine kinases to HPK1. we the EGF receptor as the receptor tyrosine kinase possesses motifs to the Grb2 domain with and also the Shc docking protein which Grb2 P. S. T. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. I. T. 1994; Google Scholar). EGF stimulation of transfected Cos1 cells induced the recruitment of HPK1 into a stable complex with both the autophosphorylated EGF receptor and The of HPK1 with the EGF receptor not HPK1 kinase activity but by Grb2 and that Grb2 HPK1 to specific Of interest, HPK1 in EGF with the that HPK1 is into a complex with the activated HPK1 also tyrosine in Cos1 cells activated of the receptor the Src cytoplasmic tyrosine kinases, which are in hematopoietic cells S. A. EMBO J. PubMed Scopus Google Scholar, T. 1995; PubMed Scopus Google Scholar). is possible that hematopoietic receptor tyrosine kinases and which Grb2 through Shc may interact with HPK1. is of to that such as and factor can activation of the pathway in hematopoietic cell which potentially might Ste20-like kinases such as HPK1. In the of the factor receptor, a specific is for activation but is in dispensable for activation Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). These results suggest a in which SH2/SH3 adaptors may HPK1 to proteins in hematopoietic cells and signaling is are that be transfected HPK1 is in the pathway, and we have been to EGF stimulation the effect of HPK1 the that HPK1, Ste20 may have multiple and and is not interactions HPK1 tyrosine phosphorylation control HPK1 regulation of the stress-activated MAPK is also possible that in hematopoietic HPK1 may proteins Grb2. In is of that a SH2/SH3 adaptor protein closely related to Grb2 has been R. J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The we have the in vitro and in interactions of the HPK1 proline-rich motifs with proteins can as the for the binding and biological activators of HPK1 in hematopoietic cell In we have that the Ste20-related kinase HPK1 has a C-terminal tail with proline-rich motifs that are closely related to of and synergistically to the Grb2 SH3 In transfected HPK1 is to activated EGF receptor and Shc and following EGF These the that Grb2 might both to the pathway and to a Ste20-related kinase, the of cross-talk between pathways by distinct serine/threonine kinases. The biological and in of HPK1 are are to Mayer for the of of Grb2.