Tyrosine phosphorylation of protein phosphatase 2A in response to growth stimulation and v-src transformation of fibroblasts.
The catalytic subunit of protein phosphatase 2A (PP2A) is inactivated by in vitro phosphorylation of Tyr307 by receptor and nonreceptor protein tyrosine kinases (Chen, J., Martin, B. L., and Brautigan, D. L. (1992) Science 257, 1261-1264). Here we show the phosphorylation of PP2A in cells under different growth conditions. In lysates of nontransformed murine 10T1/2 fibroblasts, there were two forms of PP2A at 36 kDa detected after two-dimensional gel electrophoresis and immunoblotting with anti-PP2A peptide antibody. These two forms exactly comigrated with unphosphorylated purified PP2A and the PP2A 32P-labeled by in vitro phosphorylation with p60v-src kinase. The phosphorylated form of PP2A recovered from red blood cells or produced by in vitro phosphorylation was eliminated by incubation with tyrosine-specific phosphatase (PTP1B). Transformation of 10T1/2 cells by expression of p60v-src resulted in most of the PP2A in the cells being converted to a phosphorylated form that was reactive with anti-phosphotyrosine antibody. Serum starvation of cells reduced the amount of phosphorylated PP2A, whereas serum stimulation of quiescent cells caused an increase to the same relative amount of phosphorylated PP2A as in src-transformed cells. Addition of epidermal growth factor to quiescent NeoR cells (10T1/2 fibroblasts overexpressing epidermal growth factor receptors) temporarily increased the level of phosphorylation of PP2A, with a peak at 5-15 min and a return to basal level within 60 min. The results show that PP2A is phosphorylated in intact cells, and the extent of this modification is increased by growth factors or cell transformation, providing evidence for a physiological mechanism of PP2A regulation.
Chen
J
J
Section of Biochemistry, Brown University, Providence, Rhode Island 02912.
Parsons
S
S
Brautigan
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D016391:Genes, src; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D054648:Protein Phosphatase 2; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000906:Antibodies; D003470:Culture Media; D046911:Macromolecular Substances; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2