Mapping of the p56lck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro.
The protein tyrosine kinase p56lck and other members of the src family can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p21ras, is a substrate of p56lck. Here, tryptic peptides of p56lck-phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56lck phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activating activity versus p21ras was then tested using a p21ras-dependent GTPase assay system. Our results demonstrate that p56lck-mediated tyrosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p21ras.
Department of Biology, F. Hoffmann-La Roche Inc., Basel, Switzerland.
K E
KE
Amrein
B
B
Panholzer
J
J
Molnos
N A
NA
Flint
J
J
Scheffler
H W
HW
Lahm
W
W
Bannwarth
P
P
Burn
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)