Regulation of protein kinase cascades by protein phosphatase 2A

The MAP kinase pathway is activated by a wide variety of external signals leading to cell proliferation or differentiation. However, it is not clear whether activation of this pathway is required for cellular responses or whether it is only one branch point in signal transduction. To investigate these questions, we generated constitutively activated and interfering mutants of MAP kinase kinase 1. The activated mutants stimulated PC12 cell neuronal differentiation and transformed NIH 3T3 cells. The interfering mutants inhibited growth factor-induced PC12 differentiation, growth factor stimulation of proliferation, and reverted v-src- and ras-transformed cells. These results therefore show that, depending on cellular context, activation of MAP kinase kinase is necessary and sufficient for cell differentiation or proliferation.

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Mitogenic stimulation of cells induces rapid and transient activation of MAP kinases. Here we report that a growth factor-inducible gene, 3CH134, encodes a dual specificity phosphatase that dephosphorylates and inactivates p42MAPK both in vitro and in vivo. In vitro, 3CH134 protein dephosphorylates both T183 and Y185 in p42MAPK. In serum-stimulated normal fibroblasts, the kinetics of inactivation of p42MAPK coincides with the appearance of newly synthesized 3CH134 protein, and the protein synthesis inhibitor cycloheximide leads to persistent activation of MAP kinase. Expression of 3CH134 in COS cells leads to selective dephosphorylation of p42MAPK from the spectrum of phosphotyrosyl proteins. 3CH134 blocks phosphorylation and activation of p42MAPK mediated by serum, oncogenic Ras, or activated Raf, whereas the catalytically inactive mutant of the phosphatase, Cys-258-->Ser, augments MAP kinase phosphorylation under similar conditions. The mutant 3CH134 protein also forms a physical complex with the phosphorylated form of p42MAPK. These findings suggest that 3CH134 is a physiological MAP kinase phosphatase; we propose the name MKP-1 for this phosphatase.