Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858 -6861). Additional library screening and 5 rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to ␣-and ␤-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to ␣-and ␤-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to ␣-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of ␣-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (␣-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that ␣-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with ␣-adducin. We propose naming this adducin homologue ␥-adducin.Protein kinase Cs are a heterogeneous group of phospholipiddependent kinases important for cell growth and differentiated functions (reviewed in Ref. 1). The family can be divided into three categories based on enzymatic properties. The conventional or Group A PKCs 1 are calcium-dependent kinases whose activities are stimulated by diacylglycerol or phorbol esters. The novel or Group B PKCs are calcium-independent but still diacylglycerol-stimulatable. The atypical or Group C PKCs are calcium-and diacylglycerol-independent. Most cells express more than one type of PKC, which implies that PKCs have unique rather than overlapping functions.Activation of Group A and B PKCs is regulated by receptormediated production of diacylglycerol through phospholipase C or D pathways (1). In many cases, activation correlates with PKC redistribution from soluble to particulate fractions. However, in other cases, evidence for activation in the absence of measurable translocation has been noted (2-5). The correlation ...

Previously, we showed caveolae contain a population of protein kinase Cα (PKCα) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCα and measured the interaction of recombinant PKCα with these membranes. PKCα bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCα-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCα binding. A 100–amino acid sequence from the middle of sdr competitively blocked PKCα binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.

We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with protein kinase C (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858 -6861). In this paper, we report a novel sequence, clone 72, isolated according to this method. Clone 72 has a 4.8-kilobase pair open reading frame; antibodies to clone 72 recognize a >200-kDa protein in cell and tissue extracts. Clone 72 message and protein are detected in a variety of tissues. Immunoprecipitation studies demonstrate that clone 72 is the major >200-kDa binding protein described previously in REF52 fibroblasts (Hyatt, S. L., Liao, L., Aderem, A., Nairn, A., and Jaken, S. Protein kinase C (PKC)1 is a family of phospholipid-dependent kinases involved in basic cellular functions, including regulation of growth, differentiation, and gene expression (1, 2). The role of individual PKCs in these processes is not yet known; however, since most cells express more than one type of PKC, it seems likely that individual PKCs have unique rather than overlapping functions. All of the PKCs require phosphatidylserine for maximal activity; however, PKCs can be grouped according to differences in their dependence on other activators. In addition to phosphatidylserine, conventional PKCs require calcium and diacylglycerol, novel PKCs require only diacylglycerol, and atypical PKCs require nothing more. Several other lipid modifiers of PKC activity have also been identified, and there is some evidence that they may selectively influence individual isozyme activities (1). Selective isozyme activation in response to physiological agonists has been noted and may be a result of the recognized differences in cofactor requirements among the PKCs (3-6).In addition to isozyme selective activation, isozyme-specific functions may depend on selective substrate recognition. However, only minor differences in substrate specificity among the isozymes have been observed in in vitro assays (7,8). Recently, immunofluorescence studies have demonstrated unique subcellular localizations for individual PKCs (9 -11) (data not shown). Thus, targeting of individual PKCs to specific subcellular addresses may be a means of restricting accessibility to substrates and, thereby, provide the mechanism for isozyme-selective phosphorylation events in vivo. To isolate proteins that interact with PKCs with high affinity, we developed an assay for identifying PKC-binding proteins (12-14). Subsequently, this assay was adapted to screen expression libraries and isolate cDNA clones for PKC-binding proteins (15). Binding proteins isolated according to this strategy are also substrates (16). In this manuscript, we report the full-length sequence of one of these binding proteins, clone 72. The results indicate that clone 72 is widely expressed and is a major PKC-binding protein in REF52 fibroblasts. MATERIALS AND METHODSLibrary Screening-A gt11 REF52 cDNA library was prepared and screened for PKC-binding proteins as described in Ref. 15. Positive colonies were pla...

Protein kinase C (PKC) plays a major role in regulating cell growth, transformation, and gene expression; however, identifying phosphorylation events that mediate these responses has been difficult. We expressioncloned a group of PKC-binding proteins and identified a high molecular weight, heat-soluble protein as the major PKC-binding protein in REF52 fibroblasts (Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C., and Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422). In this study, we demonstrate that this PKC-binding protein, clone 72, is also a PKC substrate in vitro and in vivo. Using a combination of phosphopeptide mapping, Edman degradation, and electrospray mass spectrometry, serine residues 283, 300, 507, and 515 were identified as the major in vitro PKC phosphorylation sites in clone 72. Phosphorylation state-selective antibodies were raised against phosphopeptides encompassing each of the four phosphorylation sites. These antibodies were used to determine that phorbol esters stimulate phosphorylation of serines 283, 300, 507, and 515 in cultured cells, indicating that clone 72 is directly phosphorylated by PKC in living cells. Phosphorylated clone 72 preferentially accumulates in membrane protrusions and ruffles, indicating that PKC activation and clone 72 phosphorylation are involved in membrane-cytoskeleton remodeling. These data lend further evidence to the model that PKCs directly interact with, phosphorylate, and modify the functions of a group of substrate proteins, STICKs (substrates that interact with C-kinase). Protein kinase C (PKC)1 is a family of phospholipid-dependent protein kinases expressed in all cells and tissues (1). PKC activity has been associated with a variety of growth and pathological defects, indicating that PKCs are involved in regulating fundamental cellular processes. PKC is the major cellular receptor for tumor-promoting phorbol esters, and consequently, PKC activation has been linked to later events in multistage carcinogenesis, i.e. tumor promotion/progression. Phorbol esters and other PKC activators rapidly induce a variety of cellular responses including cell shape changes, cytoskeletal remodeling, decreased cell-cell communication, and increased exocytosis. The diversity of the observed responses has made it complicated to focus on particular phosphorylation events that mediate changes in biological activity. Identifying target proteins and determining how PKC phosphorylation alters their activities are keys to understanding the role of PKC in cell regulation.Several approaches have been used to identify PKC-binding proteins. PICKs (proteins that interact with C-kinase) are PKC substrates identified by yeast two-hybrid screening (2). RACKs (receptors for activated C-kinase) are non-substrate proteins that bind to catalytically active PKC. These may target active PKCs to the vicinity of appropriate substrate proteins (3). Several years ago, we began using a PKC overlay assay to identify PKC-interacting proteins (4 -6). We found that many of the bind...

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.

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We have used a blot overlay assay to detect protein kinase C (PKC) interactions with other proteins. In many cases, the PKC binding proteins are also PKC substrates [Chapline et al. (1993) J. Biol. Chem. 268, 6858]. The purpose of the current studies was to characterize the PKC domains involved in the interactions with other proteins. alpha, beta, and epsilon isoforms of PKC interact with the same binding proteins in fibroblast cell extracts. These results indicate that constant rather than isozyme-specific (variable) regions are the major determinants of the interactions studied. PKC binding required phosphatidylserine (PS), indicating that the PS binding regulatory domain of PKC is involved in the interactions. The PKC pseudosubstrate peptide sequence, which is contained within the regulatory domain, also showed PS-dependent binding to the PKC binding proteins. To further investigate the role of the pseudosubstrate peptide in promoting PKC-protein interactions, an N-terminal truncation mutant lacking the pseudosubstrate sequence was prepared. Binding of the mutant alpha-PKC was diminished compared to wild-type alpha-PKC, although some binding was still apparent. These results indicate that the pseudosubstrate sequence contributes to, but is not the sole determinant of, PKC binding activity.