14-3-3 INHIBITS THE DICTYOSTELIUM MYOSIN-II HEAVY-CHAIN-SPECIFIC PROTEIN-KINASE-C ACTIVITY BY A DIRECT INTERACTION - IDENTIFICATION OF THE 14-3-3 BINDING DOMAIN

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isola ted from Dictyostelium discoideum, regulates myosin II assembly and lo calization in response to the chemoattractant cyclic AMP. Immunoprecip itation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14-3-3 protein (Dd14-3-3). This protein has recently been implicat ed in the regulation of intracellular signaling pathways via its inter action with several signaling proteins, such as PKC and Raf-l kinase. We demonstrate that the mammalian 14-3-3 zeta isoform inhibits the MHC -PKC activity in vitro and that this inhibition is carried out by a di rect interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14-3 -3 in the cytosol in a cyclic AMP-dependent manner, whereas the membra ne-bound active MHC-PKC was not found in a complex with Dd14-3-3. This suggests that Dd14-3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14-3-3 as well as 14-3-3 zeta through its C1 doma in, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-l kinase. Our ex periments thus show an in vivo function for a member of the 14-3-3 fam ily and demonstrate that MHC-PKC interacts directly with Dd14-3-3 and 14-3-3 zeta through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.