Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk.

The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.

[1]  J. Adams,et al.  Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p. , 2003, Biochemistry.

[2]  Y. Sanejouand,et al.  Building‐block approach for determining low‐frequency normal modes of macromolecules , 2000, Proteins.

[3]  J. Adams.  Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? , 2003, Biochemistry.

[4]  J. Shaffer,et al.  Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk. , 2001, Biochemistry.

[5]  T. Pawson,et al.  Proteins with SH2 and SH3 domains couple receptor tyrosine kinases to intracellular signalling pathways. , 1993, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

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

[7]  J. Adams,et al.  Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives , 2000, Protein science : a publication of the Protein Society.

[8]  Esa T Mikkola,et al.  Conserved hydrophobicity in the SH2–kinase linker is required for catalytic activity of Csk and CHK , 2003, FEBS letters.

[9]  L. Johnson,et al.  Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product. , 1995, Structure.

[10]  Ina Ruck,et al.  USA , 1969, The Lancet.

[11]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

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

[13]  D Cowburn,et al.  Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis. , 2001, Journal of molecular biology.

[14]  A. Tatosyan,et al.  Kinases of the Src family: structure and functions. , 2000, Biochemistry. Biokhimiia.

[15]  Wenqing,et al.  Three-dimensional structure of the tyrosine kinase cSrc , 2022 .

[16]  Tatosyan Ag,et al.  Kinases of the Src family: structure and functions. , 2000 .

[17]  T Pawson,et al.  Src homology region 2 domains direct protein-protein interactions in signal transduction. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Anderson,et al.  Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis. , 2000, Biochemistry.

[19]  Tirion,et al.  Large Amplitude Elastic Motions in Proteins from a Single-Parameter, Atomic Analysis. , 1996, Physical review letters.

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

[21]  Sheraz Yaqub,et al.  Release from Tonic Inhibition of T Cell Activation through Transient Displacement of C-terminal Src Kinase (Csk) from Lipid Rafts* , 2001, The Journal of Biological Chemistry.

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

[23]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[24]  P. Cole,et al.  Protein tyrosine kinases Src and Csk: a tail's tale. , 2003, Current opinion in chemical biology.

[25]  D. Sem,et al.  Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives. , 2002, Biochemistry.

[26]  Y. Sanejouand,et al.  Conformational change of proteins arising from normal mode calculations. , 2001, Protein engineering.

[27]  J. Eccleston,et al.  Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. , 1991, Biochemistry.

[28]  H. Varmus,et al.  Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src , 1990, Molecular and cellular biology.

[29]  Nguyen-Huu Xuong,et al.  Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with magnesium-ATP and peptide inhibitor , 1993 .

[30]  T. Hiratsuka.  New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. , 1983, Biochimica et biophysica acta.

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

[32]  J. Onuchic,et al.  Nonlinear elasticity, proteinquakes, and the energy landscapes of functional transitions in proteins , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[33]  D. Fesquet,et al.  Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism. , 1997, Biochemistry.

[34]  Kenneth A. Johnson,et al.  1 Transient-State Kinetic Analysis of Enzyme Reaction Pathways , 1992 .

[35]  J. Brugge,et al.  Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src , 1992, Molecular and cellular biology.

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

[37]  J. Frank,et al.  Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Virgil L. Woods,et al.  Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS). , 2002, Journal of molecular biology.

[39]  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.