Characterization of two activated mutants of human pp60c-src that escape c-Src kinase regulation by distinct mechanisms.
Two activated transforming mutants of human pp60c-src were found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitro conditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of "Src family" kinases.
Bjorge
J D
JD
Department of Medical Biochemistry, University of Calgary, Canada.
Bellagamba
C
C
Cheng
H C
HC
Tanaka
A
A
Wang
J H
JH
Fujita
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D019061:src-Family Kinases
D000952:Antigens, Polyomavirus Transforming; D010710:Phosphates; D010748:Phosphopeptides; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C000631346:CSK protein, human