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Little is known about differential expression, functions, regulation, and targeting of "atypical" protein kinase C (aPKC) isoenzymes in vivo. We have cloned and characterized a novel cDNA that encodes a Caenorhabditis elegans aPKC (PKC3) composed of 597 amino acids. In post-embryonic animals, a 647-base pair segment of promoter/enhancer DNA directs transcription of the 3.6-kilobase pair pkc-3 gene and coordinates accumulation of PKC3 protein in ϳ85 muscle, epithelial, and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion, and reproduction. Mammalian aPKCs promote mitogenesis and survival of cultured cells. In contrast, C. elegans PKC3 accumulates in non-dividing, terminally differentiated cells that will not undergo apoptosis. Thus, aPKCs may control cell functions that are independent of cell cycle progression and programmed cell death. PKC3 is also expressed during embryogenesis. Ablation of PKC3 function by microinjection of antisense RNA into oocytes yields disorganized, developmentally arrested embryos. Thus, PKC3 is essential for viability. PKC3 is enriched in particulate fractions of disrupted embryos and larvae. Immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/ plasma membrane at the apical surface of intestinal cells and in embryonic cells. A candidate anchoring/ targeting protein, which binds PKC3 in vitro, has been identified.

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...

Association of an atypical protein kinase C (aPKC) with an adapter protein can affect the location, activity, substrate specificity, and physiological role of the phosphotransferase. Knowledge Atypical protein kinase C (aPKC) 1 isoforms, which include mammalian PKCs and and Caenorhabditis elegans PKC3 (1-3), are involved in transmitting mitogenic, inflammatory, and anti-apoptotic signals; regulating gene expression; controlling vesicular trafficking and ion channel activities; establishing cell polarity; and mediating asymmetric cell divisions (4 -12). To exert control over such diverse aspects of cell physiology, aPKCs regulate effector proteins in cytoplasm, cytoskeleton, nucleus, and at the surfaces of plasma and internal membranes (4 -14). aPKCs lack structural features that mediate direct association with cytoskeleton or organelles. 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. The aPKC "recruitment" model further suggests that systematic characterization of aPKC adapter proteins will ultimately yield novel insights into regulatory properties, substrate specificities, and precise physiological roles of atypical PKCs.The nematode C. elegans is an attractive model system for studies on aPKC adapter proteins. C. elegans physiology is regulated by signaling molecules, mechanisms and pathways that are operative in mammals (21, 22). Only one aPKC (PKC3) is encoded by the C. elegans genome (3). PKC3 is expressed and anchored at all developmental stages (3). This Ca 2ϩ -and diacylglycerol (DAG)-independent kinase is essential for the progression of embryogenesis, asymmetry in early cell divisions, and overall viability of the organism (3, 11). In 1-cell embryos, ϳ25% of PKC3 associates with Par-3, a multi-PDZ domain protein that is crucial for generating intracellular polarity (11,12). Mechanisms governing formation of PKC3⅐Par-3 complexes, the identity of targets for bound PKC3, and the precise biochemical/physiological function of the complex remain to be defined. Association of Ͼ90% of PKC3 with cytoskeleton/membranes in embryos indicates that additional adapter proteins are expressed during early phases of C. elegans development (3). In post-embryonic C. elegans, PKC3 accumulates in a highly asymmetric fashion in intestinal, pharyngeal, and other cells (3). Polarized enrichment of PKC3 in nondividing cells that will not undergo apoptosis suggests that anchored PKC3 plays distinct roles in terminally differentiated cells. By using k...