Regulation of the Nonreceptor Tyrosine Kinase Brk by Autophosphorylation and by Autoinhibition

Abstract

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parametersKm and kcat both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family. Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parametersKm and kcat both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family. epidermal growth factor nickel-nitrilotriacetic acid human embryonic kidney matrix-assisted laser desorption ionization Src homology Brk is a nonreceptor tyrosine kinase that was identified in a study of kinase expression in human metastatic breast tumors (1Mitchell P.J. Barker K.T. Martindale J.E. Kamalati T. Lowe P.N. Page M.J. Gusterson B.A. Crompton M.R. Oncogene. 1994; 9: 2383-2390PubMed Google Scholar). Brk expression was low or undetectable in normal mammary tissues or in benign lesions. However, approximately two-thirds of the breast tumors that were examined expressed significant levels of Brk, and 27% of the tumors overexpressed Brk by ≥5-fold (one tumor displayed a 43-fold increase in Brk expression) (2Barker K.T. Jackson L.E. Crompton M.R. Oncogene. 1997; 15: 799-805Crossref PubMed Scopus (131) Google Scholar). Expression of Brk in cultured mammary epithelial cells makes them more sensitive to the mitogenic effects of EGF,1 and increases anchorage-independent proliferation (3Kamalati T. Jolin H.E. Mitchell P.J. Barker K.T. Jackson L.E. Dean C.J. Page M.J. Gusterson B.A. Crompton M.R. J. Biol. Chem. 1996; 271: 30956-30963Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Brk expression can also partially transform NIH3T3 fibroblasts (3Kamalati T. Jolin H.E. Mitchell P.J. Barker K.T. Jackson L.E. Dean C.J. Page M.J. Gusterson B.A. Crompton M.R. J. Biol. Chem. 1996; 271: 30956-30963Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These results suggest that inappropriate expression of Brk may contribute to the development of breast cancer. Brk is usually considered to be a member of a separate family of cytoplasmic tyrosine kinases (the Frk family) that includes Frk, Brk, Srms, and Sik, the mouse ortholog of Brk (1Mitchell P.J. Barker K.T. Martindale J.E. Kamalati T. Lowe P.N. Page M.J. Gusterson B.A. Crompton M.R. Oncogene. 1994; 9: 2383-2390PubMed Google Scholar, 4Robinson D.R., Wu, Y.M. Lin S.F. Oncogene. 2000; 19: 5548-5557Crossref PubMed Scopus (847) Google Scholar, 5Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3143) Google Scholar, 6Vasioukhin V. Serfas M.S. Siyanova E.Y. Polonskaia M. Costigan V.J. Liu B. Thomason A. Tyner A.L. Oncogene. 1995; 10: 349-357PubMed Google Scholar). Brk is more distantly related to the Src family kinases (there is 46% amino acid identity between Brk and c-Src) (1Mitchell P.J. Barker K.T. Martindale J.E. Kamalati T. Lowe P.N. Page M.J. Gusterson B.A. Crompton M.R. Oncogene. 1994; 9: 2383-2390PubMed Google Scholar, 4Robinson D.R., Wu, Y.M. Lin S.F. Oncogene. 2000; 19: 5548-5557Crossref PubMed Scopus (847) Google Scholar). Unlike Src family kinases, Brk lacks an N-terminal consensus sequence for acylation and membrane association. Furthermore, the genomic structure of Brk is quite distinct from that of Src kinases, suggesting that Brk has diverged significantly from the Src kinases in evolution (7Mitchell P.J. Barker K.T. Shipley J. Crompton M.R. Oncogene. 1997; 15: 1497-1502Crossref PubMed Scopus (39) Google Scholar). Brk possesses sequences that are predicted to form Src homology 3 (SH3) and Src homology 2 (SH2) domains (1Mitchell P.J. Barker K.T. Martindale J.E. Kamalati T. Lowe P.N. Page M.J. Gusterson B.A. Crompton M.R. Oncogene. 1994; 9: 2383-2390PubMed Google Scholar). These domains bind to proline-rich sequences and to phosphotyrosine-containing sequences, respectively (8Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2228) Google Scholar, 9Kuriyan J. Cowburn D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 259-288Crossref PubMed Scopus (468) Google Scholar). Three-dimensional structures of the Src family kinases Src and Hck (10Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1249) Google Scholar, 11Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar, 12Schindler T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 13Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (221) Google Scholar) indicate that the SH3 and SH2 domains bind to other regions of the kinase in an intramolecular manner. The SH3 domain binds a polyproline type II helix in the linker region between the SH2 domain and the catalytic domain. The SH2 domain binds a sequence in the C-terminal tail that requires phosphorylation on Tyr-527 by c-Src kinase (Csk) (14Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1085) Google Scholar). These intramolecular interactions stabilize an inactive (or “down-regulated”) conformation of the enzyme, and SH3 and SH2 ligands can potently activate Src kinases (15Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (538) Google Scholar, 16Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 17Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 18Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar). In addition to the negative regulatory roles of the Src SH2 and SH3 domains, they are involved in specific substrate recognition (14Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1085) Google Scholar, 19Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (340) Google Scholar, 20Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar). Binding of by the SH2 SH3 domains Src kinases to and activates the catalytic domain P. Miller W.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). are with to the and regulation of Brk. the substrate specificity of the Brk catalytic domain has not characterized and identified to Brk P.J. Crompton M.R. Oncogene. 2000; 19: PubMed Scopus Google S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google is not the SH3 and SH2 domains of Brk in an manner. Brk possesses a tyrosine residue (Tyr-447) the but is not this is or is involved in Brk regulation. In Src family kinases, of Tyr-527 (the to increases kinase and expression of a mutant in fibroblasts to (14Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1085) Google Scholar). Mutation of to the of Brk to anchorage-independent suggesting that Brk be in a Src kinases (3Kamalati T. Jolin H.E. Mitchell P.J. Barker K.T. Jackson L.E. Dean C.J. Page M.J. Gusterson B.A. Crompton M.R. J. Biol. Chem. 1996; 271: 30956-30963Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, in to a mutant of to tyrosine kinase activity overexpressed in cells S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). of Brk kinase (or of member of the Frk kinase family) has carried and to In this we of by on the purified Brk The for Brk was the of Crompton of of was to the Brk The for and The Brk was a was using the to from the were by wild-type Brk and mutant of Brk were expressed in cells using the of cells were with Brk 3 of cells were and with cells were in a in of A and was to and The was with of that in A. at for the was with of A Brk was from the with of A The was a of that in A. The was with of Brk was with A were and for tyrosine kinase activity using the were and at were by on an peptide The were purified by and characterized by matrix-assisted laser desorption ionization of mass The sequences of the M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Z. Eck M.J. Harrison S.C. M. J. S.R. M.J. Cantley L.C. Nature. 1995; 373: PubMed Scopus Google Z. Eck M.J. Harrison S.C. M. J. S.R. M.J. Cantley L.C. Nature. 1995; 373: PubMed Scopus Google Z. Eck M.J. Harrison S.C. M. J. S.R. M.J. Cantley L.C. Nature. 1995; 373: PubMed Scopus Google S. S.R. Miller W.T. Struct. Biol. 2001; 8: PubMed Scopus Google SH2 peptide M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Brk C-terminal was is Brk kinase activity was by a M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S.C. D. X. 1995; PubMed Scopus Google Scholar). were at in of 3 and experiments with Brk, the enzyme was with for at M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). were in and kinetic were by to the using for were using a of and Src peptide for peptide were using a of peptide and The activation was by of the a of using is in the of the in is the the in the of and is the of peptide M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The was using enzyme and of were using and Src peptide Brk or Brk were with in 3 and 2 at for In Brk was with in the for were by and by and mass spectrometry was carried out at the 2 of Brk or Brk was on and by Brk were from the to an with for with and at with in to the The were with in acid in an and to a was and with to mass was on a mass in the The in the mass spectrometry experiments were predicted of Brk using the M.R. I. A. J.C. 1997; PubMed Scopus Google Scholar). The was by the synthetic peptide to of in M. Sicheri F. Pico A. M. Kuriyan J. Miller W.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to in an the was with Binding were carried out by 2 of Brk (or mutant with of in and a at the was with the Brk to the peptide was in of and using The were to membrane and with and an cells were cultured in with and B. were The Brk expression was from and was a from were carried out using to the from the The cells were with of for to normal growth The cells were and cells were in 2 with for The were by at at and by with of A for at were with of and of A at The were by and to was carried out using or The were using and We a of Brk using the Sf9/baculovirus expression system. of cells were with Brk Brk expression 3 of this cells were and in a and we purified Brk by on and a The was by with and at a consistent with the mass The purified has a specific peptide were by the J.E. PubMed Scopus Google Scholar) and a S.C. D. X. 1995; PubMed Scopus Google Scholar). In the the of is to the of a in at this has the that is a The and the results in are from the We experiments with of and at of peptide substrate to the for These experiments the the for and the We also that Brk to the in the kinase not phosphorylation by peptide phosphorylation in the that the for this substrate is were using the of were carried out at enzyme and The peptide phosphorylation in the that the for this substrate is in a were using the of were carried out at enzyme and The substrate specificity for Brk has not We carried out experiments using peptide recognition for of tyrosine kinases. of the that are by nonreceptor tyrosine kinases and and that are by tyrosine kinases and Z. Eck M.J. Harrison S.C. M. J. S.R. M.J. Cantley L.C. Nature. 1995; 373: PubMed Scopus Google Scholar). The results that the substrate specificity of Brk is more closely related to the specificity of Src and the the synthetic peptide with the sequence M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) the for Brk The substrate with the sequence a the but also displayed a other peptide In of the Src and were more the tyrosine kinase The peptide with the sequence significant phosphorylation by Brk, and the was with a that of the Src peptide The of kinase catalytic domains of a N-terminal that and a C-terminal that makes most of the with substrate 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). In kinases, to the is by a the activation loop S.R. Annu. Rev. 2000; PubMed Scopus Google Scholar). in Src kinases, the activation loop is that of peptide to the catalytic is T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Autophosphorylation of increases the catalytic activity of the enzyme by the activation loop to out of the to Brk a tyrosine at a within the kinase domain that a (1Mitchell P.J. Barker K.T. Martindale J.E. Kamalati T. Lowe P.N. Page M.J. Gusterson B.A. Crompton M.R. Oncogene. 1994; 9: 2383-2390PubMed Google Scholar). We carried out experiments to Brk is by within the activation we Brk with and the by and These experiments that Brk is of of the enzyme with and the of Brk a tyrosine the (Tyr-447) that play a role in intramolecular we carried out phosphorylation experiments in the of the enzyme that the C-terminal in Src kinases. not significant phosphorylation of Brk in experiments not We carried out mass spectrometry experiments to sites on Brk. We Brk purified from and Brk an The were on and the were and with with and by mass spectrometry We on of Brk that were in the and that were by the mass of a in the The a in has a mass that to a peptide from the activation loop of Brk. peptide and other and in to and The sequence is between Brk and the in Src is not in Src (the residue is an is to be the of in Brk, we the involvement of We also additional and that the in mass the of were in These are from the N-terminal region of Brk. We in the were not in in the We not for phosphorylation at in the of Brk. To the of the at we a mutant form of Brk in was to mutant was expressed in cells and purified by the for wild-type Brk. We also expressed and purified other Brk a mutant in the C-terminal regulatory and a mutant at both the and We the activity of the The Y342A and but with and by The activity of the mutant was with that of wild-type Brk In of the enzyme with and the of The catalytic activity of most kinases is by phosphorylation within the activation We the of on activity for wild-type Brk and for the In of wild-type Brk a increase in enzyme a synthetic peptide substrate The Y342A mutant form of Brk a specific activity wild-type Brk, consistent with a role for this residue in kinase activation of the Y342A mutant with and in increase in kinase activity The mutant form of Brk a specific activity wild-type Brk in this suggesting that may in play a role in Brk autoinhibition. Autophosphorylation of the mutant not increase the the mutant displayed a of activity that was between wild-type Brk and the Y342A in the of the other of the mutant not to increase enzyme activity experiments that Brk activity is by at but by an In the form of Src kinases, intramolecular of the SH2 and SH3 domains enzyme activity F. Kuriyan J. Struct. Biol. 1997; PubMed Scopus Google Scholar). of of interactions in the activation loop and enzyme activation (15Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (538) Google Scholar, 16Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 17Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 18Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar). We that of synthetic peptide ligands for the SH3 and SH2 domains of Src kinases activate the by the (15Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (538) Google Scholar, M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the mutant form of Brk displayed an kinase we for the of synthetic SH3 and SH2 ligands to activate Brk and Brk. We SH2 a phosphotyrosine-containing peptide that the consensus sequence for to the Src SH2 M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and a phosphotyrosine-containing peptide that to the C-terminal sequence of Brk We the peptide in the study the consensus sequence for to the Brk SH2 domain is and we that the of Brk a for the SH2 domain. However, we were to Brk activity in the of of not In the of we a increase in kinase activity We also a proline-rich peptide that binds to the SH3 domains of Src family kinases V. V. Miller W.T. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar) an of Brk. peptide was to activate wild-type Brk with a for of is with the of for this peptide an of Src kinases V. V. Miller W.T. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). is that the SH3 domain of Brk a role in enzyme regulation. We carried out experiments with the mutant of Brk. The proline-rich peptide a of additional activation in the mutant of Brk for intramolecular interactions the SH2 domain of Brk, we the of the of the SH2 domain. In we the of wild-type Brk, Y342A Brk, Brk, and Brk to bind to an for the SH2 domain We that Src kinases not bind to peptide the of this Src kinases at Tyr-527 bind M. Sicheri F. Pico A. M. Kuriyan J. Miller W.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Brk to the SH2 The Y342A a increase in was a significant increase in for the and and suggesting that of the C-terminal tyrosine residue increases the of the SH2 domain. We for Brk in We the of Brk wild-type Brk, and is a residue in the SH3 and the is predicted to interactions with polyproline sequences P.J. Crompton M.R. Oncogene. 2000; 19: PubMed Scopus Google Scholar). a negative we also cells with Brk a at the of the were expressed at levels with wild-type Brk we for a of in Brk activity The results show that the and kinase activity wild-type Brk, with the of To for Brk was from and by with the mutant The Y342A mutant a of consistent with on purified Brk The and displayed a activity wild-type the of activity These results suggest that the and the normal intramolecular regulation of Brk and increase the catalytic activity of Brk. Brk purified from we carried out experiments to the substrate and regulation of this We peptide substrate specificity of the Brk catalytic domain using that are specific for tyrosine kinases and The specific for nonreceptor tyrosine kinases were to specific for kinases The peptide the of the However, the peptide the that is an of catalytic was the the recognition Z. Eck M.J. Harrison S.C. M. J. S.R. M.J. Cantley L.C. Nature. 1995; 373: PubMed Scopus Google Scholar). phosphorylation of this peptide was by a with the other The results suggest in addition to the kcat a role in the of specific from by Brk. The results also indicate that be that the of the Src peptide and the kcat of the The synthetic peptide in on the specificity of the Brk catalytic domain. However, in recognition is to also on interactions with other regions of Brk. Src kinases, in the are to by SH3 SH2 domain interactions (14Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1085) Google Scholar, P. Miller W.T. PubMed Google Scholar). The polyproline or in activate the Src kinases by and the substrate to the enzyme, The that Brk on regions for substrate recognition A mutant form of Brk with a SH3 domain phosphorylation of the P.J. Crompton M.R. Oncogene. 2000; 19: PubMed Scopus Google Scholar). in the SH2 domain of Brk to with and a of The most in was in experiments with a in that the sequence of binds to on mass spectrometry suggest that in the activation loop or in the N of Brk be possible sites for Sik, the mouse ortholog of Brk, also on domain interactions for substrate phosphorylation of the S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). Furthermore, the proline-rich region of was to be the for to the SH3 domain of Brk or We that Brk is by in the of and the enzyme mass spectrometry experiments specific of the activation most at the to the in Brk also to additional sites in the N at The of is not at but they may for the phosphorylation the Y342A mutant was expressed in the Y342A mutant form of Brk we a but significant increase in the activity of Brk with and The in activity for Brk in to kinases, an increase of is at using the M. T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). kinases are to the by of in of the and peptide sites by the activation loop S.R. Annu. Rev. 2000; PubMed Scopus Google Scholar). We considered the that the Brk purified from cells was on that additional phosphorylation not a in To address this we Brk with the tyrosine and the The form of Brk the increase in enzyme activity not on Brk expressed in cells not the enzyme is by In a mutant of Brk a to anchorage-independent growth (3Kamalati T. Jolin H.E. Mitchell P.J. Barker K.T. Jackson L.E. Dean C.J. Page M.J. Gusterson B.A. Crompton M.R. J. Biol. Chem. 1996; 271: 30956-30963Abstract Full Text Full Text PDF PubMed Scopus (105) Google a mutant of tyrosine kinase activity overexpressed in cells S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). results on Brk of that is involved in enzyme The mutant of Brk specific activity to wild-type synthetic peptide the mutant an to bind to an for the SH2 suggesting that an was in the mutant and expressed in the mutant form of Brk in tyrosine kinase activity and a of We also to activate Brk by addition of SH2 domain but activation was and at of The ligands in this were a peptide specific for the Src SH2 domain and a the Brk C-terminal is that the Brk SH2 domain has a specificity from that of Src S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, Z. J. P. Pawson T. M. T. Baltimore D. S. Cantley L.C. Mol. Cell. Biol. 1994; PubMed Scopus Google and a be more in Brk. results to a role for in Brk and are consistent with the results on expression of S. X. V. T. Tyner A.L. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). We that the in activity for Brk (3Kamalati T. Jolin H.E. Mitchell P.J. Barker K.T. Jackson L.E. Dean C.J. Page M.J. Gusterson B.A. Crompton M.R. J. Biol. Chem. 1996; 271: 30956-30963Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) be the of an with interactions between Brk and other that are for We that the SH3 domain of Brk also a role in enzyme regulation A peptide activates Brk with a that is to that for Src kinases, the of the SH3 domain in enzyme regulation has The mutant form of Brk also kinase activity wild-type Brk expressed in cells that is not in is the Brk we in cells the form of the on Src and was to the with to the of the The identity of the kinase for C-terminal phosphorylation of Brk is we were to Brk by with We not phosphorylation of by mass spectrometry in the or the of Brk. is that the interactions be more are for of the form of Brk. The the to the activity and regulation of Brk using purified other Frk family nonreceptor tyrosine kinase has in the results may also shed light on the structure and biochemical of other of this We Crompton for the Brk