Identification of N-terminal lobe motifs that determine the kinase activity of the catalytic domains and regulatory strategies of Src and Csk protein tyrosine kinases.

Csk and Src protein tyrosine kinases are structurally homologous but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory Src homology 3 (SH3) and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this study, we identified structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the alpha-helix C region, a beta turn between the beta4 and beta5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with one another to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase.

[1]  R. Yacobi,et al.  Autoinhibition of Bcr-Abl through Its SH3 Domain , 2003, Molecular Cell.

[2]  G. Superti-Furga,et al.  Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase , 2003, Cell.

[3]  S. Hubbard,et al.  Protein tyrosine kinase structure and function. , 2000, Annual review of biochemistry.

[4]  W. Muller,et al.  Activation of Src family kinases in Neu-induced mammary tumors correlates with their association with distinct sets of tyrosine phosphorylated proteins in vivo. , 1995, Oncogene.

[5]  Xiaofeng Lin,et al.  A new strategy to produce active human Src from bacteria for biochemical study of its regulation. , 2006, Biochemical and biophysical research communications.

[6]  P. Cole,et al.  Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail. , 2001, Biochemistry.

[7]  M. Mann,et al.  The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species. , 1996, European journal of biochemistry.

[8]  Satoru Takeuchi,et al.  Structure of the Carboxyl-terminal Src Kinase, Csk* , 2002, The Journal of Biological Chemistry.

[9]  G. Superti-Furga,et al.  Structural Coupling of SH2-Kinase Domains Links Fes and Abl Substrate Recognition and Kinase Activation , 2008, Cell.

[10]  Jonathan A. Cooper,et al.  Tyr527 is phosphorylated in pp60c-src: implications for regulation. , 1986, Science.

[11]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[12]  K. Parang,et al.  Docking-based Substrate Recognition by the Catalytic Domain of a Protein Tyrosine Kinase, C-terminal Src Kinase (Csk)* , 2006, Journal of Biological Chemistry.

[13]  Benoît Roux,et al.  The N-terminal end of the catalytic domain of SRC kinase Hck is a conformational switch implicated in long-range allosteric regulation. , 2005, Structure.

[14]  Michael J. Eck,et al.  Three-dimensional structure of the tyrosine kinase c-Src , 1997, Nature.

[15]  P. Cole,et al.  Domain interactions in protein tyrosine kinase Csk. , 1999 .

[16]  G. Superti-Furga,et al.  The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions. , 1997, Journal of molecular biology.

[17]  J. Kuriyan,et al.  High yield bacterial expression of active c‐Abl and c‐Src tyrosine kinases , 2005, Protein science : a publication of the Protein Society.

[18]  Y. Takayama,et al.  Transmembrane Phosphoprotein Cbp Positively Regulates the Activity of the Carboxyl-terminal Src Kinase, Csk* , 2000, The Journal of Biological Chemistry.

[19]  Osamu Miyashita,et al.  Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk. , 2004, Journal of molecular biology.

[20]  John Kuriyan,et al.  Structural Basis for the Recognition of c-Src by Its Inactivator Csk , 2008, Cell.

[21]  D O Morgan,et al.  Cyclin-dependent kinases: engines, clocks, and microprocessors. , 1997, Annual review of cell and developmental biology.

[22]  D. Fabbro,et al.  The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. , 2005, Structure.

[23]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[24]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

[25]  John Kuriyan,et al.  Crystal structure of the Src family tyrosine kinase Hck , 1997, Nature.

[26]  J. Kuriyan,et al.  Activation of the Sire-family tyrosine kinase Hck by SH3 domain displacement , 1997, Nature.

[27]  Toshifumi Takao,et al.  Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases , 2000, Nature.

[28]  P. Jeffrey,et al.  Structural basis of cyclin-dependent kinase activation by phosphorylation , 1996, Nature Structural Biology.

[29]  G. Sun,et al.  Mutations in the N-terminal regulatory region reduce the catalytic activity of Csk, but do not affect its recognition of Src. , 1999, Archives of biochemistry and biophysics.

[30]  J. Kuriyan,et al.  Structures of Src-family tyrosine kinases. , 1997, Current opinion in structural biology.

[31]  Keykavous Parang,et al.  Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Yuehao Wang,et al.  Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase. , 2006, Journal of molecular biology.

[33]  Gongqin Sun,et al.  Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation , 1998, Oncogene.

[34]  Philip D. Jeffrey,et al.  Crystal structure of the p27Kip1 cyclin-dependent-kinase inibitor bound to the cyclin A–Cdk2 complex , 1996, Nature.

[35]  M. Lamers,et al.  Structure of the protein tyrosine kinase domain of C-terminal Src kinase (CSK) in complex with staurosporine. , 1999, Journal of molecular biology.

[36]  Xin-Yun Huang,et al.  Src Tyrosine Kinase Is a Novel Direct Effector of G Proteins , 2000, Cell.

[37]  John Kuriyan,et al.  An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor , 2006, Cell.

[38]  Jonathan A. Cooper,et al.  The when and how of Src regulation , 1993, Cell.

[39]  Xiaofeng Lin,et al.  Subdomain switching reveals regions that harbor substrate specificity and regulatory properties of protein tyrosine kinases. , 2007, Biochemistry.

[40]  P. Cole,et al.  Peptide and protein phosphorylation by protein tyrosine kinase Csk: insights into specificity and mechanism. , 1998, Biochemistry.

[41]  Wei Wang,et al.  Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src. , 2002, Archives of biochemistry and biophysics.

[42]  Osamu Miyashita,et al.  Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation. , 2005, Journal of Molecular Biology.