Molecular Dynamics Characterization of the C2 Domain of Protein Kinase Cβ

Abstract

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of ␤PKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACKlike sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of ␤PKC. ␤PKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. ions. The results show that calcium stabilizes the ␤-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACKbinding site and the pseudo-RACK site are not notably modified by the removal of Ca 2؉ ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of ␤PKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca 2؉ ions are able to interact with two molecules of O-phospho-L-serine. These data suggest that Ca 2؉ ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation. Protein kinase C (PKC)1 is a family of protein kinases that undergo translocation from one intracellular compartment to another when activated by neurotransmitters, hormones, and growth factors. Most members of this family are activated by phosphatidylserine (PS), diacylglycerol (DG), and, to different extents, by other lipid second messengers and Ca 2ϩ ions (1, 2). There are at least three subfamilies of PKCs, classified according to their homology and sensitivity to activators (2-4). ␤PKC belongs to the so-called classic PKC subfamily, or cPKC (␣, ␤I, ␤II, and ␥ kinases). The members of this class contain four conserved domains (C1, C2, C3, and C4) inter-spaced with isozyme-unique (variable or V) domains. The ␤I and ␤II PKC isozymes are splice products of the same gene and therefore differ only in their C-terminal V5 domain (5).The C2 domain is found also in proteins other than PKC (4). Because many of these proteins bind lipids and particularly PS in a calcium-dependent manner, it was obvious to suggest that the calcium-and PS-binding sites reside within this domain (6). Sequence alignment studies (4) revealed that the C2 domains exhibit two types of topologies (type I and II), but all have a ␤-sandwich fold composed of four ␤-strands in each face of the structure (7). Based on this homology, the C2 domain was suggested to contain the calcium switch required for localization to the membrane (8). However, immunofluorescence studies did not agree with simple localization of the activated PKC isozymes to the plasma membrane. We found that activated PKC isozymes are each l...

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

“…The V1 domain of ⑀PKC is homologous to the C2 domain of the ␤PKC (1). However, there is an additional RACK-binding site in the V5 region of ␤PKC (38) and molecular dynamics studies with the C2 region of ␤PKC showed that an intramolecular interaction between the RACK and the RACK-binding site in the C2 region is not possible (39). Instead we suggest that the intramolecular interaction between the RACK and the RACK-binding site in ␤PKC is likely to occur between the C2 and V5 regions in ␤PKC.…”

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

“…In its Ca 2ϩ -bound state, this protein fold mediates an electrostatic interaction with acidic phospholipids, phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (55,56), and translocates the kinase to the plasma membrane (47). In addition, Ca 2ϩ binding induces conformational changes in the regulatory domain of the kinase (47), thereby affecting its binding properties toward protein ligands (57). Therefore, our results indicate that PKC binding to filamin may be regulated by fluctuations of the local Ca 2ϩ concentration at the plasma membrane.…”

Lipoxygenases and their metabolites in formation of plant stress tolerance

“…The similarity of the amino acid sequence and topology typical of polycystine-1, lipoxygenase and α-toxin enabled to unite these proteins in the PLAT-domain family that is related to the С 2 family [17,18]. Most of proteins, whose composition includes the С 2 -domain, are involved in the transduction of signals and membrane traffic including proteins that are implicated in the production of lipid secondary messengers (phospholipase А 2 , phospholipase С s , phosphatidilinositole-3-kіnase and in phosphorylation of other proteins (protein kinase С) [19][20][21]. That is the reason why LOX is referred to as "signal" enzyme that executes catalysis in the state associated with membrane structures [16,19,22].…”