A Novel Adapter Protein Employs a Phosphotyrosine Binding Domain and Exceptionally Basic N-terminal Domains to Capture and Localize an Atypical Protein Kinase C

Abstract

Atypical protein kinase C isoforms (aPKCs) transmit regulatory signals to effector proteins located in the cytoplasm, nucleus, cytoskeleton, and membranes. Mechanisms by which aPKCs encounter and control effector proteins in various microenvironments are poorly understood. By using a protein interaction screen, we discovered two novel proteins that adapt a Caenorhabditis elegans aPKC (PKC3) for specialized (localized) functions; protein kinase C adapter 1 (CKA1, 593 amino acids) and CKA1S (549 amino acids) are derived from a unique mRNA by alternative utilization of two translation initiation codons. CKA1S and CKA1 are routed to the cell periphery by exceptionally basic N-terminal regions that include classical phosphorylation site domains (PSDs). Many hormones and growth factors elicit activation of phospholipases that produce diacylglycerol (DAG) 1 (1, 2). Protein kinase C (PKC) isoenzymes disseminate signals carried by DAG. Amplification and routing of signals are achieved because classical (␣, ␤I, ␤II, ␥) and novel (␦, ⑀, , , ) PKC isoforms (cPKCs and nPKCs, respectively) are DAG-dependent Ser/Thr phosphotransferases that translocate from cytoplasm to sites in membranes where DAG accumulates in response to hormones (1-4). Activated PKCs phosphorylate proteins that control secretion, mitogenesis, cytoskeleton organization, gene transcription, and many other physiological processes (1-3, 5-9).Atypical PKCs (aPKCs), which include vertebrate PKC and PKC isoforms and Caenorhabditis elegans PKC3 (1, 4, 8), also regulate critical cell functions. PKC and/or PKC activate the Ras-mitogen-activated protein kinase cascade, stimulate gene transcription, inhibit apoptosis, modulate ion channel activities, phosphorylate nucleolin in the nucleus, and mediate translocation of a glucose transporter between internal and plasma membranes (9 -16). C. elegans PKC3 is required for polarized accumulation of several regulatory proteins along portions of the periphery of 1-cell embryos (17, 18). Thus, aPKCs apparently regulate effector proteins at multiple intracellular locations.Mechanisms by which aPKCs are activated and targeted to specific microenvironments are poorly understood. All PKCs have C-terminal catalytic domains and N-terminal pseudosubstrate sites and Cys-rich regions (C1 domains) (1, 2, 4); phosphatidylserine stimulates catalytic activity of all PKCs. However, aPKCs do not bind Ca 2ϩ , DAG, or phorbol esters (which mimic DAG) and are not activated or translocated by these molecules (19 -21). aPKCs also lack transmembrane domains, cytoskeleton attachment sites, and organelle targeting motifs. Unlike other PKC isoforms, aPKCs escape endosome-mediated degradation when cells are incubated chronically with hormones, DAG analogs, or phorbol esters (19 -21). Thus, the paradigm of DAG-mediated membrane translocation/activation and subsequent degradation cannot explain targeting and regulation of aPKCs.Recent evidence suggests a "recruitment model" for incorporation of aPKCs into signaling pathways. aPKCs exhibit sub-* This wo...

Caenorhabditis elegans num-1Negatively Regulates Endocytic Recycling

“…We sequenced cDNA clone yk269a11 (kindly provided by Y. Kohara) and confirmed the existence of the 1.2-kb transcript, which we have termed num-1B ( Figure 1A). Analysis of this transcript by RT-PCR showed that, like the 3.1-kb transcript characterized by Zhang et al (2001a), the num-1B transcript is transpliced to SL1. The B transcript is predicted to encode a protein of 401 amino acids that is not significantly similar in sequence to any other proteins in the public databases.…”

“…These two proteins result from the use of two distinct initiator methionines at positions 1 and 44 in the open reading frame of the longer protein (Zhang et al 2001a) ( Figure 1A). Both contain the PTB domain found in Numb proteins from other species, which has been shown to mediate protein-protein interactions ( Figure 1P) (Uhlik et al 2005).…”

“…Transcripts from the num-1 locus: A cDNA derived from the C. elegans Numb locus, num-1 (previously called cka-1; Zhang et al 2001a), has been shown to encode two proteins of 75 and 64 kDa, both of which show strong sequence similarity to Numb proteins from both mice and Drosophila (Zhang et al 2001a). These two proteins result from the use of two distinct initiator methionines at positions 1 and 44 in the open reading frame of the longer protein (Zhang et al 2001a) ( Figure 1A).…”

Structural Properties and Mechanisms That Govern Association of C Kinase Adapter 1 with Protein Kinase C3 and the Cell Periphery

“…Thus, elucidation of alternative mechanisms that enable aPKCs to encounter and control effector proteins in discrete microenvironments is an important goal. Recent investigations (reviewed in our companion paper (45)) suggest that aPKC functions are diversified and specialized via interactions with adapter proteins (6, 14 -20). Candidate adapter proteins possess two fundamental features: a tethering domain that ligates an aPKC, and a distinct targeting region that routes the adapter⅐aPKC complex to intracellular sites enriched in substrate-effector proteins and/or regulatory molecules that modulate phosphotransferase activity.…”

“…A yeast two-hybrid interaction screen yielded a unique C. elegans cDNA that encodes two novel PKC3-binding proteins (45). These proteins, which were named protein kinase C adapter 1 (CKA1) and CKA1S, are expressed throughout the life span of C. elegans and accumulate near the inner surface of plasma membranes in vivo and in transfected cells (45).…”