14–3-3 Inhibits theDictyosteliumMyosin 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 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 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 through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation

“…It is therefore likely that the downstream effects of PKC⑀ are mediated by the C1 domains. These structures have in several studies been shown to exert different biological effects (Lehel et al, 1995;Pawelczyk et al, 1998;Kiss et al, 1999;Aroca et al, 2000) and to interact with other proteins (Matto-Yelin et al, 1997;Yao et al, 1997;Pawelczyk et al, 1998;Hausser et al, 1999;Johannes et al, 1999).…”

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

“…Our findings do not exclude the possibility that Myo2 is phosphorylated at other sites by protein kinases other than those in the septation pathway. In Dictyostelium, myosin II is phosphorylated by a myosin heavy chain specific protein kinase C (Matto-Yelin et al, 1997) and the fission yeast protein kinase C homolog Pck2 localizes to the division site at cytokinesis (Sayers et al, 2000). Myo2 is not regulated by phosphorylation of conserved sites in its essential light chain Cdc4 and the regulatory light chain Rlc1 lacks the serine at amino acid position 19 that is associated with the regulation of Myo2 function in nonmuscle cells (Bresnick, 1999).…”

Expression of 14-3-3 ζ and interaction with protein kinase C in the rat retina in early diabetes

“…Many investigators have reported that 14-3-3s act as potential regulators of PKC, which is controlled by the interactions between them [10][11][12][13]. However, the effects of 14-3-3 on PKC have been controversial, showing conflicting results of inhibition or activation [5,9,13], and the biological significance of this event remains unclear.…”

“…However, the effects of 14-3-3 on PKC have been controversial, showing conflicting results of inhibition or activation [5,9,13], and the biological significance of this event remains unclear. In the present study, we sequentially confirmed decreased 14-3-3 ζ levels, up-regulated PKC and less interaction between the two in the diabetic retina.…”

“…Moreover, this isotype is present in significant amounts in the retina, as well as in the brain, and is necessary for light adaptation processes or differentiation in photoreceptors [7,8]. It is well established that 14-3-3 proteins function as a protein kinase C (PKC) regulator [9][10][11][12][13]. In vitro, 14-3-3 ζ has been reported to be an endogenous PKC inhibitor [5,14], but the role of 14-3-3 ζ in the diabetic retina remains unclear.…”