Molecular Analysis of the Interactions between Protein Kinase C-ε and Filamentous Actin

Abstract

Protein kinase C-epsilon (PKC-⑀) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-⑀ and actin. Full-length recombinant PKC-⑀, but not PKC-␤II, -␦, -, or -, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-⑀ amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-⑀ or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-⑀ colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-⑀ phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-⑀ sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-⑀ in a catalytically active conformation.

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

“…The PKC⑀ binding to and activation by F-actin have been shown to be blocked by the peptide LKKQET (Prekeris et al, 1998), which is identical to the structure that was removed in PKC⑀ without actinbinding site. We investigated whether introduction of this peptide would interfere with the PKC⑀ pathway leading to neurite outgrowth.…”

“…The interaction with F-actin was shown to stabilize PKC⑀ in an open conformation in vitro (Prekeris et al, 1998). The decreased neurite-inducing capacity of PKC⑀ upon deletion of the actin-binding site may therefore be due to the fact that a closed conformation of PKC⑀ will be favored and the neurite-inducing domains may consequently be hidden by the catalytic domain.…”

“…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). To investigate the importance of this motif for the effect of PKC⑀ on neurite outgrowth, we created expression vectors encoding both EGFP-fused full-length PKC⑀ and PKC⑀ RD lacking the ABS, ⑀FL⌬ABS and ⑀RD⌬ABS ( Figure 3A).…”

“…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).…”

Molecular mechanisms of mechanosensing in muscle development

“…Apart from Enigma family proteins, other Z-disk-associated proteins were identified as potential anchors of PKC. Actin binds PKC under specific physiological conditions and preferentially anchors certain activated PKC isoforms (Prekeris et al, 1996(Prekeris et al, , 1998. In addition, a PKC-binding protein that colocalizes with Z-disks functions as a receptor for activated PKC-kinase (RACK) (Robia et al, 2001(Robia et al, , 2005.…”