title: 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.
abstract: Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation 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 implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 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 direct 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 membrane-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 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14-3-3 family 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.
authors.affiliation: Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
authors.forename: M
authors.initials: M
authors.lastname: Matto-Yelin
authors.forename: A
authors.initials: A
authors.lastname: Aitken
authors.forename: S
authors.initials: S
authors.lastname: Ravid
mesh_terms: D048948:14-3-3 Proteins; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004023:Dictyostelium / Q000187:drug effects* / Q000201:enzymology*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D018995:Myosin Heavy Chains; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D018995:Myosin Heavy Chains