Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts.
Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [gamma-32P]ATP and [gamma-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occurred at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min. The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phosphorylation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular weight of about 100 000 and the ATP-specific main component a molecular weight of 110 000. The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002472:Cell Transformation, Viral; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D013539:Simian virus 40
D008565:Membrane Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium