Targeting of protein kinase Calpha to caveolae.

Previously, we showed caveolae contain a population of protein kinase Calpha (PKCalpha) 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 PKCalpha and measured the interaction of recombinant PKCalpha with these membranes. PKCalpha 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 PKCalpha-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCalpha binding. A 100-amino acid sequence from the middle of sdr competitively blocked PKCalpha 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.

Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification.

Human and rabbit paraoxonases/arylesterases were purified to homogeneity by chromatographic and gel electrophoretic/isofocusing procedures coupled with activity stains. N-terminal and peptide sequence analysis suggested retention of the secretion signal sequence and allowed design of oligonucleotide probes. The probes were used to isolate a 1294-bp rabbit paraoxonase cDNA clone, which, in turn, was used to isolate three human cDNA clones. Comparison of rabbit and human protein and cDNA sequences indicated a high degree of sequence conservation (approximately 85% identity) and verified that paraoxonase retains its signal sequence (except for the N-terminal Met). The rabbit cDNA encodes a protein of 359 amino acids and the human a protein of 355 amino acids. In situ hybridization demonstrated, as expected, that the paraoxonase gene maps to the long arm of human chromosome 7. Arginine at position 192 specifies high activity paraoxonase and glutamine low activity human paraoxonase. Variation in protein levels explains the variation of enzyme activity observed within a genetic class. Toxicity studies showed that raising rat plasma paraoxonase levels by i.v. administration of partially purified rabbit paraoxonase protected animals against cholinesterase inhibition by paraoxon and chlorpyrifos oxon. Protection correlated with the relative rates of hydrolysis of these two compounds.

Cell cultures and retroviral particles from a tumor of a moray eel.

Until recently, fish cell culture primarily has been useful only in the propagation and study of epidemic viruses significant to the fishing industry. Such fish cell lines derived were developed by appropriating classical techniques of mammalian cell culture, with serum as the major growth supplement. Using an approach in which culture medium is formulated in a cell-type-specific manner with minimal serum and a variety of synergistic supplements, several fish cell lines have been derived that may serve multiple uses. We established cell lines from a potentially tumorous skin lesion of a green moray eel (Gymnothorax funebris) and control tissues, and identified putative retroviral particles in the medium from the tumor cells that are not present in medium from cultures of normal cells from the same eel. The relationship between the virus and the cause of the tumor is not clear, but the genomic structure of this virus should provide useful information in understanding the evolution of retroviruses in general.

Interaction cloning of protein kinase C substrates.

We have previously used an overlay assay technique to detect proteins that interact with protein kinase C (PKC) (Hyatt, S. L., Klauck, T., and Jaken, S. (1990) Mol. Carcinogenesis 3, 45-53). In some cases, binding proteins were also identified as substrates. Therefore, we used the overlay assay approach to screen a rat kidney lambda gt11 cDNA library to isolate and identify additional PKC substrates. Two clones have now been characterized. 35A is the rat homologue of the myristoylated alanine-rich C kinase substrate (MARCKS)-related F52 cDNA, whereas 35H is a partial cDNA with substantial homology to the 3' end of beta-adducin. Both cDNAs encode proteins that bind phosphatidyl-serine (PS) and are substrates for PKC. Phosphorylation decreased both PS and PKC binding activities. Both proteins contain high density positive charge domains similar to that found in the major PKC substrate MARCKS. These results demonstrate that PKC interactions with certain substrate proteins are of sufficiently high affinity to facilitate their isolation via interaction cloning.

Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells.

Immunocytofluorescence studies demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells but not in their SV40-transformed derivatives [Jaken et al. (1989) J. Cell Biol. 109, 697-704; Hyatt & Jaken (1990) Mol. Carcinog. 3, 45-53]. Discrete localizations imply that PKC is targeted to these areas possibly via protein-protein interactions. We have used an overlay assay to detect alpha-PKC binding proteins. The molecular interactions between alpha-PKC and the binding proteins depended on phospholipid and either calcium or phorbol esters. Unlike the kinase activity, binding activity was detected in the absence of added calcium, indicating that calcium, which is necessary for phosphorylation of most substrates, is not required for binding. Vinculin and talin, two focal contact proteins, bound alpha-PKC. REF52 cells express several annexins (I, II, and VI) which bind PKC. Both annexin I expression and vinculin expression were decreased in SV40-REF52 cells. The two major REF52 cell binding proteins (p71 and p > 200 kDa) were also down-regulated in the transformed cells, indicating transformation-sensitive regulation of PKC binding protein activity.

Protein kinase C domains involved in interactions with other proteins.

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.

Purification of rabbit and human serum paraoxonase.

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.

A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodeling.

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 expression-cloned 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).

Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence.

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.

35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.

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 alpha-and beta-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 alpha- and beta-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 alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-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 alpha-adducin. We propose naming this adducin homologue gamma-adducin.

Identification of a major protein kinase C-binding protein and substrate in rat embryo fibroblasts. Decreased expression in transformed cells.

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. (1994) Cell Growth & Differ. 5, 495-502). Expression of clone 72 message and protein are decreased in progressively transformed REF52 cells. Since clone 72 is both a protein kinase C (PKC)-binding protein and substrate, decreased levels of clone 72 may influence both the subcellular location of endogenous PKCs as well as signaling events associated with clone 72 phosphorylation. Our results emphasize that the role of PKCs in carcinogenesis may involve several factors, including the quantity and location of the PKCs isozymes and their downstream targets.

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.