Raf kinase inhibitory protein is required for cerebellar long-term synaptic depression by mediating PKC-dependent MAPK activation.

It was demonstrated previously that a positive feedback loop, including protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), is required for the gradual expression of cerebellar long-term depression (LTD). PKC and MAPK are mutually activated in this loop. MAPK-dependent PKC activation is likely to be mediated by phospholipase A2. On the other hand, it is not clear how PKC activates MAPK. Therefore, the entire picture of this loop was not fully understood. We here test the hypothesis that this loop is completed by the PKC substrate, Raf kinase inhibitory protein (RKIP). To test this hypothesis, we used a mutant form of RKIP that is not phosphorylated by PKC and thus constitutively inhibits Raf-1 and MEK, upstream kinases of MAPK. When this RKIP mutant was introduced into Purkinje cells of mouse cerebellar slices through patch-clamp electrodes, LTD was blocked, while wild-type (WT) RKIP had no effect on LTD. Physiological epistasis experiments demonstrated that RKIP works downstream of PKC and upstream of MAPK during LTD induction. Furthermore, biochemical analyses demonstrated that endogenous RKIP dissociates from Raf-1 and MEK during LTD induction in a PKC-dependent manner, suggesting that RKIP binding-dependent inhibition of Raf-1 and MEK is removed upon LTD induction. We therefore conclude that PKC-dependent regulation of RKIP leads to MAPK activation, with RKIP completing the positive feedback loop that is required for LTD.