Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

FEBS Lett. 1995 Jan 9;357(3):290-6. doi: 10.1016/0014-5793(94)01376-c.

Abstract

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cyclic AMP / analogs & derivatives*
  • Cyclic AMP / pharmacology
  • Enzyme Activation
  • MAP Kinase Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinase Kinases*
  • Mitogen-Activated Protein Kinases*
  • PC12 Cells
  • Phosphorylation
  • Protein-Serine-Threonine Kinases*
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Rats
  • Thionucleotides / pharmacology*

Substances

  • Proto-Oncogene Proteins
  • Thionucleotides
  • 8-((4-chlorophenyl)thio)cyclic-3',5'-AMP
  • Cyclic AMP
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase 1
  • Mitogen-Activated Protein Kinase Kinases