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A Novel Non-catalytic Mechanism Employed by the C-terminal Src-homologous Kinase to Inhibit Src-family Kinase Activity

2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês

10.1074/jbc.m309865200

ISSN

1083-351X

Autores

Yuh‐Ping Chong, Terrence D. Mulhern, Hong‐Jian Zhu, Donald J. Fujita, Jeffrey D. Bjorge, John-Paul Tantiongco, Nikolaos Sotirellis, Daisy Lio, Glen M. Scholz, Heung‐Chin Cheng,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (TyrT), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating TyrT, CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic TyrT-independent mechanism because it occurs even in the absence of ATP or when TyrT of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK·Src complex exists in rat brain, and recombinant CHK·Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their TyrT and (ii) by this novel TyrT-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their TyrT is phosphorylated. Thus, the ability of the TyrT-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances. Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (TyrT), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating TyrT, CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic TyrT-independent mechanism because it occurs even in the absence of ATP or when TyrT of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK·Src complex exists in rat brain, and recombinant CHK·Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their TyrT and (ii) by this novel TyrT-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their TyrT is phosphorylated. Thus, the ability of the TyrT-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances. Src-family kinases (SFKs) 1The abbreviations used are: SFK, Src-family tyrosine kinase; CSK, C-terminal Src kinase; CHK, CSK-homologous kinase; MES, 4-morpholineethanesulfonic acid; HEK cells, human embryonic kidney cells. are non-receptor protein-tyrosine kinases that participate in many cellular functions ranging from cell growth and proliferation to memory and learning (1Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2164) Google Scholar). The kinase activity of SFKs is regulated by phosphorylation, as well as by their interaction with other cellular proteins. Among the various regulatory mechanisms, the most important are autophosphorylation of a consensus tyrosine (TyrA) 2CHK exists in three isoforms distinguishable by their molecular weight, two variants of the 56-kDa isoform (p56CHK) and one 52-kDa isoform (p52CHK) (23Chow L.M. Davidson D. Fournel M. Gosselin P. Lemieux S. Lyu M.S. Kozak C.A. Matis L.A. Veillette A. Oncogene. 1994; 9: 3437-3448PubMed Google Scholar, 36Bennett B.D. Cowley S. Jiang S. London R. Deng B. Grabarek J. Groopman J.E. Goeddel D.V. Avraham H. J. Biol. Chem. 1994; 269: 1068-1074Abstract Full Text PDF PubMed Google Scholar). In this manuscript all characterizations were made with p52CHK. The residues in Hck, Lyn, and Src are numbered in accordance to the amino acid sequence of the 56-kDa form of the mouse Hck and Lyn (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 37Lock P. Ralph S. Stanley E. Boulet I. Ramsay R. Dunn A.R. Mol. Cell. Biol. 1991; 11: 4363-4370Crossref PubMed Scopus (106) Google Scholar, 38Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar) and human c-Src (39Tanaka A. Gibbs C.P. Arthur R.R. Anderson S.K. Kung H.J. Fujita D.J. Mol. Cell. Biol. 1987; 7: 1978-1983Crossref PubMed Scopus (59) Google Scholar). In the Hck sequence the novel autophosphorylation site (Tyr-29) in the unique domain corresponds to Tyr-29; the consensus autophosphorylation site (TyrA) in the kinase domain corresponds to Tyr-388, and the conserved C-terminal regulatory tyrosine (TyrT) corresponds to Tyr-499. In the Lyn sequence, the consensus autophosphorylation site (TyrA) and the C-terminal regulatory tyrosine (TyrT) correspond to Tyr-397 and Tyr-508, respectively. In Src sequence, TyrA and TyrT correspond to Tyr-419 and Tyr-530, respectively. in the kinase domain and phosphorylation of a consensus regulatory tyrosine near the C terminus (TyrT) 2CHK exists in three isoforms distinguishable by their molecular weight, two variants of the 56-kDa isoform (p56CHK) and one 52-kDa isoform (p52CHK) (23Chow L.M. Davidson D. Fournel M. Gosselin P. Lemieux S. Lyu M.S. Kozak C.A. Matis L.A. Veillette A. Oncogene. 1994; 9: 3437-3448PubMed Google Scholar, 36Bennett B.D. Cowley S. Jiang S. London R. Deng B. Grabarek J. Groopman J.E. Goeddel D.V. Avraham H. J. Biol. Chem. 1994; 269: 1068-1074Abstract Full Text PDF PubMed Google Scholar). In this manuscript all characterizations were made with p52CHK. The residues in Hck, Lyn, and Src are numbered in accordance to the amino acid sequence of the 56-kDa form of the mouse Hck and Lyn (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 37Lock P. Ralph S. Stanley E. Boulet I. Ramsay R. Dunn A.R. Mol. Cell. Biol. 1991; 11: 4363-4370Crossref PubMed Scopus (106) Google Scholar, 38Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar) and human c-Src (39Tanaka A. Gibbs C.P. Arthur R.R. Anderson S.K. Kung H.J. Fujita D.J. Mol. Cell. Biol. 1987; 7: 1978-1983Crossref PubMed Scopus (59) Google Scholar). In the Hck sequence the novel autophosphorylation site (Tyr-29) in the unique domain corresponds to Tyr-29; the consensus autophosphorylation site (TyrA) in the kinase domain corresponds to Tyr-388, and the conserved C-terminal regulatory tyrosine (TyrT) corresponds to Tyr-499. In the Lyn sequence, the consensus autophosphorylation site (TyrA) and the C-terminal regulatory tyrosine (TyrT) correspond to Tyr-397 and Tyr-508, respectively. In Src sequence, TyrA and TyrT correspond to Tyr-419 and Tyr-530, respectively. (2Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar, 3Cheng H.C. Bjorge J.D. Aebersold R. Fujita D.J. Wang J.H. Biochemistry. 1996; 35: 11874-11887Crossref PubMed Scopus (19) Google Scholar). Autophosphorylation of TyrA leads to activation of SFKs (1Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2164) Google Scholar, 4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 5Sotirellis N. Johnson T.M. Hibbs M.L. Stanley I.J. Stanley E. Dunn A.R. Cheng H.C. J. Biol. Chem. 1995; 270: 29773-29780Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The crystal structure of the autophosphorylated kinase domain of the Src-family kinase Lck reveals that the phosphorylated TyrA (Tyr(P)A) stabilizes the active kinase domain configuration by forming ionic interactions with the conserved Arg in the catalytic loop (6Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (423) Google Scholar). We previously reported that the Src-family member Hck could undergo autophosphorylation at a novel site (Tyr-29) 2CHK exists in three isoforms distinguishable by their molecular weight, two variants of the 56-kDa isoform (p56CHK) and one 52-kDa isoform (p52CHK) (23Chow L.M. Davidson D. Fournel M. Gosselin P. Lemieux S. Lyu M.S. Kozak C.A. Matis L.A. Veillette A. Oncogene. 1994; 9: 3437-3448PubMed Google Scholar, 36Bennett B.D. Cowley S. Jiang S. London R. Deng B. Grabarek J. Groopman J.E. Goeddel D.V. Avraham H. J. Biol. Chem. 1994; 269: 1068-1074Abstract Full Text PDF PubMed Google Scholar). In this manuscript all characterizations were made with p52CHK. The residues in Hck, Lyn, and Src are numbered in accordance to the amino acid sequence of the 56-kDa form of the mouse Hck and Lyn (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 37Lock P. Ralph S. Stanley E. Boulet I. Ramsay R. Dunn A.R. Mol. Cell. Biol. 1991; 11: 4363-4370Crossref PubMed Scopus (106) Google Scholar, 38Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar) and human c-Src (39Tanaka A. Gibbs C.P. Arthur R.R. Anderson S.K. Kung H.J. Fujita D.J. Mol. Cell. Biol. 1987; 7: 1978-1983Crossref PubMed Scopus (59) Google Scholar). In the Hck sequence the novel autophosphorylation site (Tyr-29) in the unique domain corresponds to Tyr-29; the consensus autophosphorylation site (TyrA) in the kinase domain corresponds to Tyr-388, and the conserved C-terminal regulatory tyrosine (TyrT) corresponds to Tyr-499. In the Lyn sequence, the consensus autophosphorylation site (TyrA) and the C-terminal regulatory tyrosine (TyrT) correspond to Tyr-397 and Tyr-508, respectively. In Src sequence, TyrA and TyrT correspond to Tyr-419 and Tyr-530, respectively. in the Unique domain and that autophosphorylation of Hck at Tyr-29 contributed to Hck activation (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, the structural basis of activation by Tyr-29 autophosphorylation is not yet known. In contrast to the activating effect of TyrA and Hck Tyr-29 autophosphorylation, TyrT phosphorylation results in inactivation (7Laudano A.P. Buchanan J.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 892-896Crossref PubMed Scopus (26) Google Scholar). Crystal structures of TyrT-phosphorylated c-Src (8Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1249) Google Scholar) and Hck (9Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar) reveal that the inactive configuration of the kinase domain is stabilized by intramolecular interactions involving binding of (i) the Tyr(P)T to the SH2 domain and (ii) the SH2 kinase linker to the SH3 domain. The consensus TyrT of SFKs is thought to be phosphorylated exclusively by two upstream regulatory tyrosine kinases, C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) 2CHK exists in three isoforms distinguishable by their molecular weight, two variants of the 56-kDa isoform (p56CHK) and one 52-kDa isoform (p52CHK) (23Chow L.M. Davidson D. Fournel M. Gosselin P. Lemieux S. Lyu M.S. Kozak C.A. Matis L.A. Veillette A. Oncogene. 1994; 9: 3437-3448PubMed Google Scholar, 36Bennett B.D. Cowley S. Jiang S. London R. Deng B. Grabarek J. Groopman J.E. Goeddel D.V. Avraham H. J. Biol. Chem. 1994; 269: 1068-1074Abstract Full Text PDF PubMed Google Scholar). In this manuscript all characterizations were made with p52CHK. The residues in Hck, Lyn, and Src are numbered in accordance to the amino acid sequence of the 56-kDa form of the mouse Hck and Lyn (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 37Lock P. Ralph S. Stanley E. Boulet I. Ramsay R. Dunn A.R. Mol. Cell. Biol. 1991; 11: 4363-4370Crossref PubMed Scopus (106) Google Scholar, 38Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar) and human c-Src (39Tanaka A. Gibbs C.P. Arthur R.R. Anderson S.K. Kung H.J. Fujita D.J. Mol. Cell. Biol. 1987; 7: 1978-1983Crossref PubMed Scopus (59) Google Scholar). In the Hck sequence the novel autophosphorylation site (Tyr-29) in the unique domain corresponds to Tyr-29; the consensus autophosphorylation site (TyrA) in the kinase domain corresponds to Tyr-388, and the conserved C-terminal regulatory tyrosine (TyrT) corresponds to Tyr-499. In the Lyn sequence, the consensus autophosphorylation site (TyrA) and the C-terminal regulatory tyrosine (TyrT) correspond to Tyr-397 and Tyr-508, respectively. In Src sequence, TyrA and TyrT correspond to Tyr-419 and Tyr-530, respectively. (2Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar, 10Chow L.M.L. Jarvis C. Hu Q.L. Nye S.H. Gervais F.G. Veillette A. Matis L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4975-4979Crossref PubMed Scopus (62) Google Scholar, 11Klages S. Adam D. Class K. Fargnoli J. Bolen J.B. Penhallow R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2597-2601Crossref PubMed Scopus (100) Google Scholar, 12Kuo S.S. Moran P. Gripp J. Armanini M. Phillips H.S. Goddard A. Caras I.W. J. Neurosci. Res. 1994; 38: 705-715Crossref PubMed Scopus (37) Google Scholar). CSK is ubiquitously expressed in all mammalian tissues, whereas the expression of CHK is much more restricted; it is expressed predominantly in neurons and hemopoietic cells. Extensive biochemical evidence indicates that CSK inactivates SFKs primarily by phosphorylating their TyrT. Although there are several pieces of preliminary evidence suggesting that CHK can also phosphorylate TyrT of several SFK members, including c-Src, Lyn, Lck, and Fyn (10Chow L.M.L. Jarvis C. Hu Q.L. Nye S.H. Gervais F.G. Veillette A. Matis L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4975-4979Crossref PubMed Scopus (62) Google Scholar, 11Klages S. Adam D. Class K. Fargnoli J. Bolen J.B. Penhallow R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2597-2601Crossref PubMed Scopus (100) Google Scholar), CHK-mediated SFK inactivation remains largely uncharacterized. Using Hck, Lyn, and Src as model targets, we investigated the mechanism by which CHK inactivates SFKs. We discovered that in addition to inactivation of SFKs by specifically phosphorylating their TyrT, CHK could inactivate SFKs by a novel inhibitory mechanism. In this mechanism CHK directly binds to SFKs to form stable complexes, and this binding alone is sufficient to inactivate SFKs. To further confirm the validity our in vitro observations, we attempted to determine if CHK could bind and inactivate Hck in transfected HEK293T cells. Results of our experiments using the transfected HEK293T cells reveal that CHK·Hck complex formation and inactivation of Hck occurred even when TyrT of Hck was replaced by phenylalanine. The results indicate that CHK·Hck complex formation and Hck inactivation by this novel inhibitory mechanism are not mediated by binding of TyrT of Hck to the active site of CHK. Hence, this novel inhibition occurs by a non-catalytic mechanism. To support the physiological relevance of our findings, we reveal that CHK and Src co-localize to specific microdomains of rat brain plasma membrane and that CHK and Src form stable protein complex(es) in rat brain. Furthermore, we also demonstrate that the interaction can suppress SFK activity regardless of the level of autophosphorylation of TyrA. Thus, by binding to and suppressing the activity of both the less active unphosphorylated form and the fully active autophosphorylated form of SFKs, CHK is capable of providing a fail-safe mechanism to down-regulate SFK signaling under all circumstances. Materials—Recombinant wild type Hck was expressed and purified as described previously (5Sotirellis N. Johnson T.M. Hibbs M.L. Stanley I.J. Stanley E. Dunn A.R. Cheng H.C. J. Biol. Chem. 1995; 270: 29773-29780Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Sicilia R.J. Hibbs M.L. Bello P.A. Bjorge J.D. Fujita D.J. Stanley I.J. Dunn A.R. Cheng H.C. J. Biol. Chem. 1998; 273: 16756-16763Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The “kinase dead” Hck(K267M) mutant was generated by site-directed mutagenesis and purified by procedures as described previously (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The anti-CHK antibody was raised against a glutathione S-transferase fusion protein containing the C-terminal 89-amino acid segment corresponding to residues 379–467 of CHK. The polyclonal anti-CSK, anti-Hck, and anti-Tyr(P)-29 Hck antibodies were generated and purified as described previously (3Cheng H.C. Bjorge J.D. Aebersold R. Fujita D.J. Wang J.H. Biochemistry. 1996; 35: 11874-11887Crossref PubMed Scopus (19) Google Scholar, 4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The rabbit polyclonal anti-Tyr(P)A phosphospecific antibody (also called anti-Src[pY-418]) was from BIOSOURCE International. pRK7-BatK-flagC plasmid encoding the 52-kDa isoform of rat CHK (p52CHK) was kindly provided by Drs. I. Caras and S. Kuo (12Kuo S.S. Moran P. Gripp J. Armanini M. Phillips H.S. Goddard A. Caras I.W. J. Neurosci. Res. 1994; 38: 705-715Crossref PubMed Scopus (37) Google Scholar, 14Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar). Poly(Glu,Tyr), a random copolymer of glutamate and tyrosine (4:1) that is a nonspecific peptide substrate for tyrosine kinases, was purchased from Sigma. The two anti-Src monoclonal antibodies mAb327 and mAb(2Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar, 3Cheng H.C. Bjorge J.D. Aebersold R. Fujita D.J. Wang J.H. Biochemistry. 1996; 35: 11874-11887Crossref PubMed Scopus (19) Google Scholar, 4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 5Sotirellis N. Johnson T.M. Hibbs M.L. Stanley I.J. Stanley E. Dunn A.R. Cheng H.C. J. Biol. Chem. 1995; 270: 29773-29780Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 6Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (423) Google Scholar, 7Laudano A.P. Buchanan J.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 892-896Crossref PubMed Scopus (26) Google Scholar, 8Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1249) Google Scholar, 9Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar, 10Chow L.M.L. Jarvis C. Hu Q.L. Nye S.H. Gervais F.G. Veillette A. Matis L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4975-4979Crossref PubMed Scopus (62) Google Scholar, 11Klages S. Adam D. Class K. Fargnoli J. Bolen J.B. Penhallow R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2597-2601Crossref PubMed Scopus (100) Google Scholar, 12Kuo S.S. Moran P. Gripp J. Armanini M. Phillips H.S. Goddard A. Caras I.W. J. Neurosci. Res. 1994; 38: 705-715Crossref PubMed Scopus (37) Google Scholar, 13Sicilia R.J. Hibbs M.L. Bello P.A. Bjorge J.D. Fujita D.J. Stanley I.J. Dunn A.R. Cheng H.C. J. Biol. Chem. 1998; 273: 16756-16763Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar, 15Sabe H. Knudsen B. Okada M. Nada S. Nakagawa H. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2190-2194Crossref PubMed Scopus (80) Google Scholar, 16Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 323Google Scholar, 17Prior I.A. Harding A. Yan J. Sluimer J. Parton R.G. Hancock J.F. Nat. Cell Biol. 2001; 3: 368-375Crossref PubMed Scopus (457) Google Scholar) were purified and characterized as previously described (15Sabe H. Knudsen B. Okada M. Nada S. Nakagawa H. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2190-2194Crossref PubMed Scopus (80) Google Scholar). Chemical Cross-linking of Antibodies to Protein A-Sepharose—Data generated by the use of these reagents are shown in Fig. 10C. Rabbit IgG from preimmune sera, anti-CHK, and anti-CSK antibodies were chemically cross-linked to protein A-Sepharose with dimethyl pimelimidate (Sigma-Aldrich) following the procedures described by Harlow and Lane (16Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 323Google Scholar). Mutagenesis and Construction of Expression Vectors—The cDNA fragment encoding full-length CHK was retrieved from the pRK7-BatK-flagC plasmid by digestion with EcoR1 and BamH1. It was then used as template for a PCR reaction that introduced EcoR1 and BglII restriction sites, and the stop codon. The oligonucleotides used were 5′-CCG GAA TTC GCT AAG ATG CCA ACG CAA CGC-3′ (Primer 1 sense) and 5′-G ACT AGA TCT TCA GGG GTC CTG GCT CCG-3′ (Primer 2 antisense); the restriction sites are underlined, and the start and stop codons are in bold italics. The PCR product generated was cloned into the pBAK-PAK9 vector (Clontech) using procedures described previously (4Johnson T.M. Williamson N.A. Scholz G. Jaworowski A. Wettenhall R.E. Dunn A.R. Cheng H.C. J. Biol. Chem. 2000; 275: 33353-33364Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For the K221M mutation pBacPAK9-CHK plasmid was used as template for PCR-based mutagenesis. The oligonucleotides used were Primer 1 Sense, Primer 2 Antisense, and Primer 3 Antisense (5′-G TAC TTG GGA CAG AAG GTG GCC GTG ATG AAT ATA-3′); the A to T codon change is underlined in the sequence. The resultant plasmids, pBacPAK9-CHK or pBacPAK9-CHK(K221M), were sequenced to confirm that only the required mutation was introduced during the PCR procedure. Both plasmids were used to co-transfect with BAC-PAK6 baculoviral DNA into Sf9 cells to generate the recombinant CHK and CHK(K221M) baculoviruses according to manufacturer's instructions (Clontech). Purification of Recombinant CHK and Its Mutant Expressed in Sf9 Insect Cells—All purification procedures were carried out at 4 °C. One liter of CHK baculovirus-infected Sf9 cells (0.7 × 106 cells/ml) were harvested by centrifugation at 1000 × g for 5 min. The cell pellet was homogenized in lysis buffer (50 mm Tris, pH 7.0, 1% Nonidet P-40, 1 mm EDTA, 0.2 mg/ml benzamidine, 50 mm β-glycerophosphate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 5 mm sodium potassium tartrate, 1 mm p-nitrophenylphosphate, 10% glycerol, 0.1 mg/ml trypsin inhibitor, and 50 mm NaF). The supernatant was applied to a DEAE column (80-ml bed volume) pre-equilibrated with Buffer A (25 mm Hepes, pH 7.0, 0.1% Nonidet P-40, 10% glycerol, 1 mm EDTA, 0.2 mg/ml benzamidine, 10 mm β-glycerophosphate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 0.5 mm Na3VO4, and 50 mm NaF). The unbound fraction, which contained the majority of the CHK protein was then loaded onto a carboxymethyl cation exchange column (100 × 22 mm). Proteins bound to the carboxymethyl column were eluted with a 0–1 m NaCl gradient in Buffer A. Fractions containing CHK were further purified on a Superose-12 gel-filtration column equilibrated in Buffer A with 0.2 m NaCl. The CHK-containing column fractions were pooled and applied to a phosphotyrosine-agarose column (5-ml bed volume, 5–15 μmol of immobilized phosphotyrosine per ml). The column was washed with Buffer A containing 0.2 m NaCl. The bound proteins were eluted from the column with 100 mm p-nitrophenylphosphate in Buffer A. Fractions containing CHK were pooled and purified by Mono S cation exchange column chromatography. As shown in Fig. 1C, the final CHK preparation was >90% pure. The recombinant CHK(K221M) mutant was also purified using an identical procedure. Preparation of Crude Rat Brain Lysate for Detection of CHK·Src Protein Complexes—All procedures described were performed at 4 °C. Brain tissues from 10 adult rats were homogenized in 140 ml of lysis buffer. The homogenate was centrifuged twice at 40,000 × g for 40 min. The supernatant was separated into aliquots and frozen at –70 °C before immunoprecipitation analysis to generate the data shown in Fig. 10C. Fractionation of Rat Brain Membrane—This section describes Fig. 10B. All procedures were performed at 4 °C. Brain tissues from 10 adult rats were homogenized in Buffer B (0.32 m sucrose, 25 mm Tris-HCl, pH 7.0, 1 mm MgCl2, 0.2 mg/ml benzamidine, 0.1 mg/ml phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 600 × g for 10 min to remove the nuclei and large debris. The supernatant was centrifuged at 100,000 × g for 30 min to separate the plasma membrane and cytoplasm. The crude membrane was further separated into different membrane microdomains by sucrose density gradient centrifugation and treatment with Triton X-100 using a procedure adapted from Prior et al. (17Prior I.A. Harding A. Yan J. Sluimer J. Parton R.G. Hancock J.F. Nat. Cell Biol. 2001; 3: 368-375Crossref PubMed Scopus (457) Google Scholar) and Waheed et al. (18Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar). Briefly, the crude membrane was resuspended in 2 ml of 45% (w/v) sucrose in MBS solution (25 mm MES, pH 6.5, 150 mm NaCl, 0.2 mg/ml benzamidine, and 0.1 mg/ml phenylmethylsulfonyl fluoride). The suspension was loaded into a centrifuge tube and then overlaid with a MBS-sucrose gradient of 35% sucrose (2.8 ml), 30% sucrose (2.4 ml), 25% sucrose (2.4 ml), and 5% sucrose (2.4 ml). After centrifugation at 300,000 × g in the Beckman SW41 rotor for 16 h, 12 1-ml fractions were harvested from the top of the tube. Fractions 2–5 and 9–12 correspond to the low and high density membrane microdomains, respectively. To pellet the membrane in each fraction, MBS (9 ml) was added to the individual fractions, and the mixture was centrifuged at 300,000 × g for 30 min. The pellet from each fraction was treated with 0.5 ml of 1% Triton X-100 in Buffer B. The samples were again subjected to centrifugation at 300,000 × g for 15 min. The supernatant corresponds to the Triton X-100-soluble portion, whereas the pellet corresponds to the Triton X-100-insoluble portion. Aliquots of both portions were analyzed for the presence of CHK and Src by Western blotting. Assay of CHK Protein Kinase Activity—Three different substrates, poly(Glu/Tyr), Hck(K267M), and SFK C-terminal peptide were used to assay CHK activity. For poly(Glu/Tyr) phosphorylation the kinase assay mixture (25 μl) consisted of 8–12 μg of CHK, kinase assay buffer (20 mm Tris-HCl, pH 7.0, 10 mm MgCl2, 1 mm MnCl2, 50 μm Na3VO4), 2.5 mg/ml poly(Glu, Tyr), and 100 μm [γ-32P]ATP. An assay mixture without CHK was used as a blank to quantify the background radioactivity. Phosphorylation was allowed to proceed at 30 °C for 30 min. After termination of the reaction, CHK activity was determined following procedures previously described (3Cheng H.C. Bjorge J.D. Aebersold R. Fujita D.J. Wang J.H. Biochemistry. 1996; 35: 11874-11887Crossref PubMed Scopus (19) Google Scholar). The results are shown in Fig. 1B. Hck(K267M) was used as a bona fide substrate of CHK to investigate the efficiency of CHK in catalyzing TyrT phosphorylation. The reaction mixture contained kinase assay buffer, 100 μm [γ-32P]ATP, CHK (0.33 μm), and varying concentrations of Hck(K267M) (0–5.8 μm). The reaction mixture was incubated at 30 °C. After termination of the reaction, Hck(K267M) was separated by SDS-PAGE, and the gel was subjected to autoradiography. The individual protein bands corresponding to Hck(K267M) were excised, and the associated radioactivity was quantitated (Fig. 2C). An SFK C-terminal

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