Engineering Src family protein kinases with unnatural nucleotide specificity.

BACKGROUND Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field. RESULTS We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N6-(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N6-substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N6-(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N6-(benzyl) ATP on Fyn. CONCLUSIONS The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1-5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.

[1]  W. Miller,et al.  Substrate Specificities of the Insulin and Insulin-like Growth Factor 1 Receptor Tyrosine Kinase Catalytic Domains (*) , 1995, The Journal of Biological Chemistry.

[2]  B. Tao,et al.  Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. , 1992, Biotechnology advances.

[3]  T. Pawson.  Protein Modules and Signaling Networks , 2000 .

[4]  Charis Eng,et al.  Catalytic specificity of protein-tyrosine kinases is critical for selective signalling , 1995, Nature.

[5]  Yi Liu,et al.  Design of allele-specific inhibitors to probe protein kinase signaling , 1998, Current Biology.

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

[7]  J. Kuriyan,et al.  Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: Crystal structures of the complexed and peptide-free forms , 1993, Cell.

[8]  D. Kassel,et al.  Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. , 1995, Biochemistry.

[9]  Joseph Schlessinger,et al.  Structure of the FGF Receptor Tyrosine Kinase Domain Reveals a Novel Autoinhibitory Mechanism , 1996, Cell.

[10]  S. Courtneidge,et al.  Src family protein tyrosine kinases and cellular signal transduction pathways. , 1995, Current opinion in cell biology.

[11]  Kornelia Polyak,et al.  Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex , 1995, Nature.

[12]  J. Brugge,et al.  Identification of a transformation-specific antigen induced by an avian sarcoma virus , 1977, Nature.

[13]  B X Yan,et al.  Glycine Residues Provide Flexibility for Enzyme Active Sites* , 1997, The Journal of Biological Chemistry.

[14]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. , 1993, Biochemistry.

[15]  K. M. Abraham,et al.  Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59 fyn ) , 1991, Cell.

[16]  Tony Pawson,et al.  Protein modules and signalling networks , 1995, Nature.

[17]  S. Hanks,et al.  Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. , 1991, Methods in enzymology.

[18]  H. Iba,et al.  Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein , 1986, Molecular and cellular biology.

[19]  K. Shokat,et al.  Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Jove,et al.  Cell transformation by the viral src oncogene. , 1987, Annual review of cell biology.

[21]  B. Sefton,et al.  Most of the substrates of oncogenic viral tyrosine protein kinases can be phosphorylated by cellular tyrosine protein kinases in normal cells. , 1988, Oncogene research.

[22]  E. Krebs,et al.  Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. Baltimore,et al.  Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides , 1993, Nature.

[24]  R. Perlmutter,et al.  Cell type and developmental regulation of the fyn proto-oncogene in neural retina. , 1992, Oncogene.

[25]  R. Perlmutter,et al.  Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. , 1989, The New biologist.

[26]  S. Kanner,et al.  Mutational activation of pp60(c-src) leads to a tumorigenic phenotype in a preneoplastic Syrian hamster embryo cell line. , 1997, Cancer research.

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

[28]  Hiroto Yamaguchi,et al.  Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation , 1996, Nature.

[29]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[30]  M. T. Brown,et al.  Regulation, substrates and functions of src. , 1996, Biochimica et biophysica acta.

[31]  D. Baltimore,et al.  Modular binding domains in signal transduction proteins , 1995, Cell.

[32]  G. Fields,et al.  Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification. , 2009, International journal of peptide and protein research.

[33]  Charles S. Craik,et al.  Protein engineering : principles and practice , 1996 .

[34]  T. Hunter.  A thousand and one protein kinases , 1987, Cell.

[35]  Cook Mp,et al.  Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. , 1989 .

[36]  T. Hunter,et al.  Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling , 1995, Cell.

[37]  D. Lawrence,et al.  The Extraordinary Active Site Substrate Specificity of pp60c−src , 1995, The Journal of Biological Chemistry.

[38]  S. Schreiber,et al.  Solution structure of the SH3 domain of Src and identification of its ligand-binding site. , 1992, Science.

[39]  R. Perlmutter,et al.  Differential contribution of Lck and Fyn protein tyrosine kinases to intraepithelial lymphocyte development , 1997, European journal of immunology.

[40]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[41]  T. Hunter,et al.  Transforming gene product of Rous sarcoma virus phosphorylates tyrosine , 1980, Proceedings of the National Academy of Sciences.

[42]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.