Studies on a novel adapter protein that avidly binds atypical protein kinase C
Atypical protein Kinase C isoforms (aPKCs) transmit regulatory signals to effector proteins located in cytoplasm, the nucleus, cytoskeleton, plasma membrane and internal membranes. However, mechanisms by which aPKCs encounter and control target effector proteins in various microenvironments are poorly understood. A protein interaction screen was executed to identify partner proteins that adapt the sole C. elegans aPKC (PKC3) for specialized and localized functions. I discovered and characterized cDNA that encodes two novel PKC3 binding proteins: C kinase adapter I (CKA1, 593 amino acids) and CKA1S, 549 amino acids). The basic CKA1 and CKA1S proteins (pIs ≥ 9.4) are derived from a unique 2.1 kb mRNA by alternative utilization of two translation initiation codons. CKA1S and CKA1 are routed to the cell periphery by exceptionally basic (pI > 12) N terminal regions that includes one or two classical phosphorylation site domains (PSDs), respectively. High affinity binding of PKC3 by CKA1/CKA1S is mediated by a segment of 142 contiguous amino acids (residues 90--231) that constitute a phosphotyrosine binding (PTB) domain. Ligation of PKC3 is strictly dependent on the integrity of the entire PTB domain. Deletion of the N terminal targeting region and/or the novel central and C terminal portions (residues 232--593) of CKA1 did not diminish tethering of the kinase. Site-directed mutagenesis within the PTB domain revealed that two aromatic amino acids (Phe175, Phe221) play indispensable roles in creation of the PKC3 binding surface and/or stabilization of CKA1•aPKC complexes. Patterns of both CKA1 gene promoter activity and CKA1/CKA1S protein localization in vivo overlap extensively with previously established patterns of PKC3 expression and distribution. Transfection experiments demonstrated that membrane associated CKA1/CKA1S encounters and tightly sequesters PKC3 molecules in the internal milieu of intact cells. In polarized epithelial cells PKC3 adapter proteins are precisely targeted to a lateral region of plasma membrane that is involved in generating impermeable intracellular junctions. Thus, two classical structural modules (a PTB domain and a PSD) collaborate in a novel fashion in CKA1 adapter proteins to enable tethering and differential localization of PKC3. Avid ligation of a PKC isoform is a previously unappreciated and potentially important role for PTB modules that are embedded within a multitude of key signaling and scaffolding proteins.;I also discovered that a 13-residue segment (amino acids 212--224) of the V2 (linker) region of PKC3 creates a binding surface that interacts with the PTB domain of CKA1/CKA1S. Side chains of Ile214 , Asn216 and Phe219 are indispensable for high-affinity ligation of PKC3 by the hydrophobic surface of the PTB domain. The CKA1/CKA1S binding site in PKC3 contains neither phosphorylated nor unmodified Tyr. A peptide corresponding to residues 212--224 in PKC3 potently inhibits tethering of the kinase by CKA1/CKA1S and thereby provides a new tool for analyzing the regulatory and physiological significance of aPKC•adapter complexes. PKC3 and diacylglycerol-activated PKCs phosphorylate Ser 17 and Ser65 in the PSDs of CKA1. A key consequence of phosphorylation is disengagement of the adapter protein from the cell surface and subsequent translocation to cytoplasm and/or internal membranes.
Source: Dissertation Abstracts International, Volume: 61-09, Section: B, page: 4584.;Advisors: Charles S. Rubin.