Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function.

Abstract

Abstract. Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKCe and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKCe within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC-binding protein. The binding of PKCe to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKCe. Once established, this protein-protein interaction securely anchored PKCe to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKCe isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.VOLUTION has conserved a fundamental mechanism that ensures the sorting and fusion of secretory vesicles upon their delivery to appropriate destinations along the secretory pathway. In many eukaryotes, however, alternative pathways have emerged in which vesicle delivery and fusion have become rigorously regulated events that are capable of manifesting use-dependent variations in the probability of their occurrence. Glutamate exocytosis is a particularly striking example of a secretory event that, in presynaptic nerve terminals, has acquired a highly evolved mechanism for regulating the efficiency of excitatory synaptic transmission. Moreover, it has been established that use-dependent changes in the reliability (Stevens and Wang, 1994) and extent (Schulz et al., 1994) of glutamate exocytosis directly participate in certain forms of synaptic plasticity.Studies from many laboratories suggest that a conditional relationship may exist between the efficiency of glutamate release and the activity of presynaptic protein

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

“…Overexpression of PKC⑀ resulted in a highly motile and invasive phenotype in various cancer models (58,59), and its disruption caused inactivation of Rho family GTPases (60). It has also been shown that PKC⑀ can bind directly to both filamentous and globular forms of actin via its actin binding motif (61,62) or interact indirectly via integrins and scaffolding proteins such as receptors for activated protein kinase C (also known as RACK) (63,64). In murine embryonic fibroblasts, PKC⑀ was shown to phosphorylate vimentin and regulate ␤1 integrin recycling contributing to cell motility (24,25).…”

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

“…This structure is located between the C1 domains in the RD of PKC⑀ (Prekeris et al, 1996). Removal of the actin-binding site from PKC⑀ decreases its binding to F-actin in vitro but does not seem to alter other properties of the protein (Prekeris et al, 1998).…”

“…The C2 domain, in the RD, is crucial for the binding of PKC⑀ to its receptor for activated C-kinase (Mochly-Rosen and Gordon, 1998) and this domain has been used to specifically block the translocation and function of PKC⑀ (Johnson et al, 1996;Hundle et al, 1997). Furthermore, there is an actin-binding site between the C1 domains, unique for PKC⑀, which is of importance for the localization of PKC⑀ and also can mediate an F-actin-induced activation of the enzyme (Prekeris et al, 1996(Prekeris et al, , 1998. This is of interest because there is an increased association of PKC⑀ to the cytoskeleton during neuronal differentiation of PC12 cells (Brodie et al, 1999) and because PKC⑀ is enriched in the F-actin-rich growth cones of differentiating neuroblastoma cells (Fagerströ m et al, 1996).…”

Protein Kinase (PKC)-ε Interacts with the Serotonin Transporter (SERT) C-Terminal Region

“…PKC-ε induces neurite outgrowth during neuronal differentiation activated by various stimuli through interaction with actin filament [11]. The significance of the actin-binding site in the interaction with filaments has been implicated in neurotransmitter endocytosis [24].…”