Endogenous protein kinase activity was detected in the outer plasma membrane of 3T3 and SV 40 transformed 3T3 cells. When intact cells were incubated with [γ 32P]ATP, there was a transfer of [32P] phosphate into an acid insoluble product. The reaction was linear as a function of time (up to 30 min), proportional to the number of cells present, and dependent on temperature and Mg2+ concentration. The acid insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [γ 32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorylating exogenous substrates, histone, and phosvitin. The level of phosphorylation increased by 2 to 4 fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV 40 3T3 cells had from 5 to 10 fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV 40 transformed 3T3 cells.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1976|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology