Caveolin Interaction with Protein Kinase C

Abstract

Caveolar localization of protein kinase C and the regulation of caveolar function by protein kinase C are well known. This study was undertaken to examine whether caveolin subtypes interact with various protein kinase C isoenzymes using the caveolin scaffolding domain peptide. When protein kinase C-␣, -⑀, and -were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (␣, ⑀, and ) were detected in the same fraction as caveolin. The scaffolding domain peptide of caveolin-1 and -3, but not -2, inhibited the kinase activity and autophosphorylation of protein kinase C-␣ and -, but not of protein kinase C-⑀, overexpressed in insect cells. Truncation mutation studies of the caveolin-1 and -3 peptides demonstrated that a minimum of 16 or 14 amino acid residues of the peptide were required for the inhibition or direct binding of protein kinase C. Thus, the caveolin peptide physically interacted with protein kinase C and regulated its function. Further, this regulation occurred in a protein kinase C isoenzyme-dependent manner. Our results may provide a new mechanism regarding the regulation of protein kinase C isoenzyme activity and the molecular interaction of protein kinase C with its putative binding proteins.Several studies from independent laboratories have demonstrated that multiple phorbol ester-sensitive, classic protein kinase C (PKC) 1 isoenzymes are accumulated in caveolae. Enrichment of PKC-␣ was detected by immunoelectron microscopy (1) as well as by the cell fractionation technique using buoyant density gradient centrifugation (2). PKC-␤ and -␥ were also detected in caveolae as separated by the silica coating method from lung endothelial cells (3). Whether all PKC isoenzymes, including nonclassic isoenzymes, are similarly accumulated in caveolae, however, remains uncharacterized. PKC also regulates the function and formation of caveolae. Caveolin, the major structural protein of caveolae, contains a conserved consensus phosphorylation site of PKC as well as of v-Src (4). Phorbol ester treatment of the cell inhibits caveolae-mediated internalization and markedly reduces the number of caveola (1). Further, activation of PKC-␣ by phorbol esters dislocates this isoenzyme from caveolae. Thus, PKC is not only present in caveolae, but interacts functionally with caveolae.It has been suggested that caveolin by itself regulates the function of certain molecules accumulated in caveolae. Caveolin may directly interact with G protein, Src kinase, and HaRas as has been demonstrated using a short stretch of membrane proximal regions of the cytosolic amino terminus caveolin domain (or the caveolin scaffolding domain) (5-8).Further, a small peptide derived from this domain bound G protein directly and regulated its function (9). The specificity of binding caveolin to target molecules has been confirmed, using a random peptide sequence library, by identifying a common amino acid sequence motif (XXXXX or XXXXXX; is an aromatic residue), which is contained in ma...

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

“…Caveolin appears to be a receptor for PKC in COS cells. Peptides corresponding to the scaffolding domain of caveolin inhibit autophosphorylation, peptide phosphorylation, and phorbol ester binding of COS cells (37). Active PKC␣ and PKC⑀ have also been found in the caveolar fraction of cardiac myocytes in response to the phorbol ester phorbol 12-myristate 13-acetate and endothelin but not in response to the inactive phorbol ester 4␣-phorbol 12-myristate 13-acetate (38).…”

The Caveolin Scaffolding Domain Modifies 2-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor Binding Properties by Inhibiting Phospholipase A2 Activity

“…Another hypothesis is that cPLA 2 clustering with other signaling molecules inside caveolae may indirectly inactivate cPLA 2 activity. For instance, cPLA 2 can be stimulated by protein kinases such as protein kinase C (41) and mitogen-activated protein kinase (42), which are known to be inhibited by caveolin-1 (43).…”

m-Calpain Colocalizes with the Calcium-sensing Receptor (CaR) in Caveolae in Parathyroid Cells and Participates in Degradation of the CaR

“…Both calpain and PKC are expected to translocate to the plasma membrane after activation (15). Caveolar localization of PKC isoforms as well as regulation of caveolar function by PKC are well known (2,3,52,53). Calpains can cleave PKC␣, ␤, ␥, ␦, ⑀, and isoforms in vitro, but they cleave only the activated forms of PKC in living cells (15).…”