Characterization of the C-terminal domain of ras-GTPase-activating protein (ras-GAP) as substrate for epidermal growth factor receptor and p60c-src kinase.
We describe in vitro tyrosine phosphorylation of the C-terminal 334 amino acids of ras-GTPase-activating protein (ras-GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N-terminal domain of ras-GAP Tyr-460, which is considered to be the major phosphorylation site of ras-GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF-R compared to p60c-src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF-R) or increased (in case of p60c-src) phosphorylation of the C-terminal 334 amino acids of ras-GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be-independent from the presence of SH2 or SH3 domains-triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr-460.
Borowski
P
P
Institut für Medizinische Mikrobiologie und Immunologie Universitätskrankenhaus Eppendorf, Hamburg, Germany.
Kornetzky
L
L
Heiland
M
M
Roloff
S
S
Weber
W
W
Laufs
R
R
D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006657:Histones / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010047:Ovalbumin / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011107:Polylysine / Q000031:analogs & derivatives / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D019061:src-Family Kinases / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D006657:Histones; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D011107:Polylysine; D011099:Polyglutamic Acid; C030391:poly(glutamic acid-lysine); D014443:Tyrosine; D008345:Manganese; D010047:Ovalbumin; D066246:ErbB Receptors; D019061:src-Family Kinases; D014357:Trypsin; D008274:Magnesium