NERVE GROWTH-FACTOR STIMULATES A NOVEL PROTEIN-KINASE IN PC-12 CELLS THAT PHOSPHORYLATES AND ACTIVATES MITOGEN-ACTIVATED PROTEIN-KINASE KINASE (MEK)

Activation of mitogen-activated protein kinase (MAP kinase) plays an i mportant role in the cellular effects of nerve growth factor (NGF), Al though the precise pathway by which NGF activates MAP kinase is not cl ear, several enzymes have been identified that may form a linear phosp horylation cascade, in which MAP kinase is activated by MAP kinase kin ase (MEK). A key enzyme that links the ras-GTP complex to MEK is widel y believed to be the raf kinase. However, immunoprecipitation experime nts in PC-12 cells revealed that raf is not the major NGF-dependent ME K kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 559 5-5599]. We have identified a protein kinase from PC-12 cells that cat alyses both the phosphorylation and activation of MEK. This activity i s stimulated 3-fold in cells treated with NGF. The partial purificatio n on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF- stimulated protein kinases, The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and t he heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is s lightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50 000 Da. Moreover, isoelectric focusing of th e enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a K-m of II mu M, and requires Mg2+ as a co factor. Analysis of the activation of this enzyme in PC-12 cells trans fected with a dominant inhibitory mutant of p21(ras) suggests that thi s MEK kinase resides downstream of ras in the MAP kinase activation pa thway. Moreover, site-directed mutation of the residues on MEK that ar e phosphorylated by raf does not completely abrogate phosphorylation b y the MEK kinase, suggesting that this enzyme may share some phosphory lation sites with raf, but also phosphorylates MEK on other sites.