Independent Protein Kinase Research Articles

The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C.

Bruton tyrosine kinase (EC 2.7.1.112) [Btk, encoded by Btk in mice and BTK in humans (formerly known as atk, BPK, or emb)], which is variously mutated in chromosome X-linked agammaglobulinemia patients and X-linked immunodeficient (xid) mice, has the pleckstrin homology (PH) domain at its amino terminus. The PH domain of Btk expressed as a bacterial fusion protein directly interacts with protein kinase C in mast cell lysates. Evidence was obtained that Btk is physically associated with protein kinase C in intact murine mast cells as well. Both Ca(2+)-dependent (alpha, beta I, and beta II) and Ca(2+)-independent protein kinase C isoforms (epsilon and zeta) in mast cells interact with the PH domain of Btk in vitro, and protein kinase C beta I is associated with Btk in vivo. Btk served as a substrate of protein kinase C, and its enzymatic activity was down-regulated by protein kinase C-mediated phosphorylation. Furthermore, depletion or inhibition of protein kinase C with pharmacological agents resulted in an enhancement of the tyrosine phosphorylation of Btk induced by mast cell activation.

Translocation and Downregulation of Protein Kinase C Isoenzymes-α and -ε by Phorbol Ester and Bryostatin-1 in Human Keratinocytes and Fibroblasts

Protein kinase C isoenzymes can be subdivided into two classes, based on their requirement for calcium. Protein kinase C-alpha, beta I, -beta II, and -gamma are calcium dependent, whereas protein kinase C-gamma, -epsilon, -zeta, -eta, and -theta are calcium independent. We have examined the expression, translocation, downregulation, and activation of calcium-dependent and -independent protein kinase C isoenzymes in human skin keratinocytes and fibroblasts. Human keratinocytes and fibroblasts expressed protein kinase C-alpha, -delta, -epsilon, and -zeta mRNA and protein, whereas protein kinase C-eta (L) was detected only in keratinocytes. Protein kinase C-beta I, -beta II, -gamma, and -theta were not detected in either cell type. The protein kinase C activators 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1 (50 nM, for 5 min) induced translocation of protein kinase C-alpha and -epsilon cytosol to membrane in both keratinocytes and fibroblasts. 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1, for 18 h, induced complete downregulation (i.e., loss) of protein kinase C-alpha and -epsilon in keratinocytes, but only partial downregulation was observed in fibroblasts. The subcellular distribution of protein kinase C-delta, -zeta or protein kinase C-eta, in keratinocytes or fibroblasts, did not change in response to 12-0-tetradecanoylphorbol 13-acetate or bryostatin-1. These data indicate differential expression, subcellular distribution, and regulation of protein kinase C isoenzymes in human skin cells.

Expression and Regulation of Calcium Independent Protein Kinase C in NG 108-15 Cell Differentiation

Protein, mRNA and activity levels of the calcium-independent protein kinase C (nPKC) isoenzymes were examined in NG108-15 cells. Western blot analyses reveal that proliferating NG 108-15 cells express the δ, ϵ and η, but not the θ species. The atypical specie PKC ζ was also detected. Differentiation of these cells with dibutyryl cAMP was associated with increase in the levels of PKC ϵ, with no significant changes in its steady-state mRNA levels. The levels of the other isoforms were not altered by the differentiated state. Similarly, no changes in nPKC activity were discerned in either the soluble or particulate fractions when histone or other proteins were used as substrates. These data suggest that the PKC ϵ isoform may be important for the production and maintenance of the differentiated state in NG 108-15 cells.

KP-10, a novel protein kinase C substrate in intact mouse epidermal cells, is phosphorylated by novel protein kinase Cη and/or ξ

Recently this group found an endogenous substrate protein for Ca(2+)-independent novel protein kinase C (nPKC), i.e. KP-10 (pI 4.7/25,500 M(r)), in primary cultured mouse epidermal cells [Nishikawa, K. et al. (1992) Cell. Signal. 4, 757-776]. In the present study, the nPKC isozymes which phosphorylate KP-10 in these cells were determined. Western blot analysis revealed that PKC alpha, eta and zeta were present in epidermal cell 105,000 g supernatants and that the content of PKC zeta was much higher than those of PKC alpha and eta. Neither PKC beta, delta nor epsilon was detected in the 105,000 g supernatants. Phosphatidylserine and phorbol 12-myristate 13-acetate (PMA)-dependent KP-10 phosphorylating activity was immunoprecipitated by anti-PKC eta and zeta antibodies, but not by antiPKC alpha antibody. These results suggest that PKC eta and/or zeta phosphorylate KP-10 and play pivotal roles in intracellular signal pathways in intact epidermal cells.

Cloning and sequencing of isoform-specific regions of human Ca2+-independent protein kinase C (PKC)-encoding genes.

The cloning and sequencing of the isoform-specific regions of the human Ca2+-independent protein kinase C-encoding genes is described. These clones will serve as correct size probes for screening human genomic or cDNA libraries and isolating full-length clones.

Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.

The role of protein kinase C in calcium-dependent exocytosis was investigated in permeabilized rat basophilic leukaemia cells. When protein kinase C was down-regulated by phorbol myristate acetate (1 microM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 microM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 microM), cells lost the ability to secrete in response to 10 microM free Ca2+. In contrast, a short treatment (15 min) with phorbol myristate acetate, which maximally activates protein kinase C, potentiated the effects of calcium. Biochemical analysis of protein kinase C-deprived cells indicated that loss of the Ca(2+)-induced secretory response correlated with disappearance of protein kinase C-alpha. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-alpha to the membrane fraction and stimulated phospholipase C, suggesting that, in permeabilized cells, protein kinase C can be activated by calcium through generation of the phospholipase C metabolite diacylglycerol. The delta, epsilon and zeta Ca(2+)-independent protein kinase C isoenzymes were insensitive to phorbol myristate acetate-induced down-regulation and did not, as expected, translocate to the particulate fraction in response to calcium. Interestingly, secretory competence was restored in cells depleted of protein kinase C or in which protein kinase C itself was inhibited by non-hydrolysable GTP analogues, but not by GTP, suggesting that protein kinase C might regulate the ability of a G protein(s) directly controlling the exocytotic machinery to be activated by endogenous GTP.

Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells.

Rat pituitary GH4C1 cells express protein kinase C (PKC) transcripts for cPKC alpha, cPKC beta II, nPKC delta, nPKC epsilon, nPKC eta, and aPKC zeta, but not for cPKC gamma or nPKC theta. Of the transcripts produced, the nPKC epsilon isoform is the most abundant. Transfection of GH4C1 cells with an expression plasmid containing nPKC epsilon cDNA leads to the transient overexpression of cellular nPKC epsilon and confers enhanced phorbol ester binding activity. Transient expression of an inactive point mutant (nPKC epsilon K-->R) of nPKC epsilon, where Lys436 at the putative ATP-binding site is replaced with Arg, also confers elevated binding activity. However, only overproduction of the wild type in transfected cells increases the basal levels and stimulates the secretion of prolactin (PRL) by 12-O-tetradecanoylphorbol-13-acetate or thyrotropin-releasing hormone (TRH). In stable clones overexpressing nPKC epsilon, immunocytofluorescence and immunoblot experiments indicated that TRH causes the rapid translocation and down-regulation of an appreciable fraction of nPKC epsilon. Both the basal and TRH-stimulated levels of PRL secretion are clearly correlated with the expression level of nPKC epsilon but not with the TRH receptor densities in these clones. The dose dependence of TRH-stimulated secretion were similar in all cells overexpressing cPKC alpha, cPKC beta II, nPKC epsilon, and nPKC delta, but the enhancement of PRL secretion was specific for the overproduction of nPKC epsilon, no effect was found when other isozymes were overproduced. These findings clearly demonstrate that the expression level of nPKC epsilon in GH4C1 cells is rate-limiting for basal and TRH-stimulated PRL secretion, and they provide the first direct evidence that nPKC epsilon plays a key role in hormonal secretory processes.

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

Defect in Radiation Signal Transduction in Ataxia-telangiectasia

Exposure of mammalian cells to ionizing radiation causes a delay in progression through the cycle at several checkpoints. Cells from patients with ataxia-telangiectasia (A-T) ignore these checkpoint controls postirradiation. The tumour suppressor gene product p53 plays a key role at the G1/S checkpoint preventing the progression of cells into S phase. The induction of p53 by radiation is reduced and/or delayed in A-T cells, which appears to account for the failure of delay at the G1/S checkpoint. We have investigated further this defect in radiation signal transduction in A-T. While the p53 response was defective after radiation, agents that interfered with cell cycle progression such as mimosine, aphidicolin and deprivation of serum led to a normal p53 response in A-T cells. None of these agents caused breaks in DNA, as determined by pulse-field gel electrophoresis, in order to elicit the response. Since this pathway is mediated by protein kinases, we investigated the activity of several of these enzymes in control and A-T cells. Ca+2-dependent and -independent protein kinase C activities were increased by radiation to the same extent in the two cell types, a variety of serine/threonine protein kinase activities were approximately the same and anti-tyrosine antibodies failed to reveal any differences in protein phosphorylation between A-T and control cells. It is not evident what is the nature of the defect in signal transduction in A-T cells. However, it is clear that the p53 response is normal in these cells after exposure to some agents and it is mediated through protein kinase C or another serine/threonine kinase.

H7-resistant protein kinase C substrates in two-dimensional gels of proestrous rat anterior pituitary gland

The presence of Ca 2+-dependent and -independent protein kinase C (PKC) activities which were stimulated by phorbol-12,13-dibutyrate (PDBu) and sensitive to the kinase inhibitor staurosporine (IC 50 values approx. 100 nM) was demonstrated in proestrous rat anterior pituitary gland. These PDBu-induced activities were completely abolished by the PKC-specific inhibitors Ro31-8220 and GF109203X (3 μM). The Ca 2+-independent activity was more resistant (IC 50 = 61 μM) to the kinase inhibitor H7 than the Ca 2+-dependent activity (IC 50 approx. 20 μM), however, this (unusual) resistance to H7 was not observed in the brain regions, hypothalamus and hippocampus. Possible substrates for the Ca 2+-independent PKC in anterior pituitary were identified by two-dimensional gel electrophoresis and autoradiography following incubation in vitro with [ 32P]phosphate and 300 nM PDBu ± 300 nM staurosporine or 30 μM H7. The phosphorylation of six proteins (16, 16, 25, 36, 65 and 69 kDa) was found to be stimulated by PDBu and inhibited by staurosporine, but not H7, in whole tissue, and another two such phosphorylated proteins (each 76 kDa) were observed in microsomal subcellular fractions. These phosphoproteins may be substrates for an H7-resistant PKC isoform previously shown to mediate a number of cellular responses in rat anterior pituitary gland.

Differentiation-Associated Localization of nPKCμ, a Ca++-Independent Protein Kinase C, in Normal Human Skin and Skin Diseases

The expression of nPKC eta, a Ca(++)-independent isoform of protein kinase C in normal human skin, and skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma, nevus pigmentosus, and seborrheic keratosis, were examined by immunohistochemical staining using a polyclonal antibody raised against a synthetic peptide at a diverse region of the nPKC eta molecule. In normal epidermis, the strongest staining was observed in the uppermost granular layer with no staining of the spinous or basal layers. The inner layer of the intra-epidermal eccrine duct was also strongly stained. Weak staining was observed in several layers of the outer root sheath of the follicular infundibulum. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, intradermal eccrine duct, arrectores pilorum, melanocytes, Langerhans cells, fibroblasts, or blood vessels. In psoriatic skin, stained keratinocytes were distributed in the suprabasal layers with the most being observed in the uppermost layer and the least in layers closed to the basal layer. In squamous cell carcinoma, weak staining was observed in the keratotic cells around horny pearls. In the basal cell epithelioma and nevus pigmentosus, the cells were not stained, whereas in seborrheic keratosis, cells that stained were located in the granular layer. We conclude from the evidence presented above that nPKC eta is expressed in close association with epidermal differentiation in normal skin and skin diseases.

The Mechanism of Epipodophyllotoxin-Induced Thymocyte Apoptosis: Possible Role of a Novel Ca 2+-Independent Protein Kinase

The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase 1 by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca 2+) chelator, under conditions that inhibited apoptosis induced by the Ca 2+ ionophore A23187. Moreover, while strong increases in intracellular Ca 2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca 2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca 2+ and possibly requires a novel Ca 2+-independent PKC-like enzyme and another, utilized by Ca 2+ ionophores, that is at least partially dependent on increased intracellular Ca 2+.

Reconstitution of p21ras-dependent and -independent mitogen-activated protein kinase activation in a cell-free system.

In Xenopus oocytes, mitogen-activated protein (MAP) kinase can be activated by progesterone treatment or by microinjection of cyclin A, both of which lead to activation of the cdc2 protein kinase. The tyrosine kinase pp60v-src has previously been shown to accelerate progesterone-induced oocyte maturation and to increase the phosphorylation of ribosomal protein S6 by pp90rsk, most likely by activating MAP kinase. In extracts of resting oocytes, MAP kinase kinase and MAP kinase were activated by addition of pp60v-src or cyclin A. Activation by pp60v-src was blocked by a dominant-negative p21ras protein (RAST), but activation by cyclin A/cdc2 was unaffected. Thus these two pathways that converge at MAP kinase kinase but are clearly divergent upstream of a p21ras-dependent step can be studied in a cell-free system.

Activated release of membrane-anchored TGF-alpha in the absence of cytosol.

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.

Ca 2+-independent protein kinase Cs contain an amino-terminal domain similar to the C2 consensus sequence

Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation. Despite significant progress, the rational design of the C1 modulators remains challenging due to difficulties associated with structure determination of the C1-ligand complexes. Given the dearth of experimental structural data, computationally derived models have been instrumental in providing atomistic insight into the interactions of the C1 domains with PKC agonists. In this review, we provide an overview of the in silico approaches for seven classes of C1 modulators and outline promising future directions.

A Ca(2+)-independent protein kinase C from fission yeast.

A protein kinase C homologue of Schizosaccharomyces pombe, pkc1+, was isolated from a genomic library by screening with the Saccharomyces cerevisiae PKC1 probe. From its primary sequence and biochemical properties, we conclude that S. pombe pkc1+ encodes a phospholipid-activated Ca(2+)-independent protein kinase, homologous to the delta/epsilon classes of mammalian protein kinase C. Gene disruption experiments show that pkc1+ is not essential for cell viability; however, overexpression of the protein leads to an abnormal cell morphology and a block in cell separation following mitosis suggestive of a role in cell division. In vitro phosphorylation experiments reveal several potential pkc1+ substrates.

Opioid peptides activate phospholipase D and protein kinase C-epsilon in chicken embryo neuron cultures.

The mu-opioid peptide morphiceptin stimulated a Ca(2+)-independent protein kinase C (PKC-epsilon) that is expressed both in embryonic day 6 chicken telencephalon and in derived neuronal cultures. This activation was seen as a 2-fold increase in the activity and level of cytosolic PKC-epsilon and as a transient increase in membrane-associated PKC-epsilon following morphiceptin treatment. Morphiceptin did not activate phospholipase C-mediated phosphatidylinositol hydrolysis but did transiently activate (2- to 3-fold) phospholipase D (PLD), as measured by phosphatidylethanol formation in neuron cultures derived from embryonic day 6 or day 7 cerebral hemispheres. This PLD activation could provide an alternative source of diacylglycerol for the activation of PKC-epsilon and was naloxone-reversible and at least partially blocked by the tyrosine kinase inhibitor herbimycin A. Addition of phorbol 12-myristate 13-acetate stimulated both PLD and PKC-epsilon activities to a greater extent than opioids. The phorbol ester and insulin stimulation of PLD was also blocked by herbimycin. Both morphiceptin (in a naloxone-reversible manner) and phorbol ester increased phosphorylation of similar cytosolic proteins in intact cells, demonstrating a functional role for the PKC-epsilon activation by opioids. This is evidence that opioid receptors are transiently coupled to tyrosine kinase, PLD and PKC-epsilon activation and, by implication, to neuronal cell growth during brain morphogenesis.

1-dehydro-melengestrol acetate inhibits the growth and protein kinase C activity of androgen-independent Dunning rat prostatic tumors.

Androgen-independent Dunning R3327-AT3 rat prostate tumors are considered an appropriate model of advanced prostate cancer in humans. We recently reported that the progestational steroid melengestrol acetate (MGA) inhibited growth of these tumors on oral administration but also induced a marked involution of adrenals and androgen target organs (prostate, seminal vesicles, and testes). We report herein that the 1-dehydro derivative of melengestrol acetate (dMGA) fed to rats for 21 days also inhibited the growth of Dunning AT3 tumors by approximately 55% without causing a significant regression of adrenals or androgen-dependent tissues. Thus, tumor-growth inhibition was induced by dMGA in the absence of glucocorticoid activity. Cytosolic AT3 tumor fractions obtained by diethylaminoethyl (DEAE)-Sephacel batch chromatography were assayed for lipid- and Ca(2+)-dependent (PKC) and -independent protein kinase activities. Prostatic cytosols had equivalent activity levels of both types of kinases (approximately 2 nmol gamma-[32P]-adenosine 5'-triphosphate (ATP) incorporated mg protein-1 min-1. The PKC activity recovered from the cytosol of untreated AT3 tumors was approximately 4 times higher. Oral administration of dMGA reduced this activity by > 95%. The relationship between protein-kinase activity levels and dMGA-induced growth inhibition of androgen-independent tumors in this animal model is discussed.

The impact of molecular biology on understanding renal signal transduction.

The information that cells require to grow and prosper comes from diverse sources, adjacent to them and from considerable distances. Through evolution, cells have developed multiple mechanisms to acquire and process information. When a signaling mechanism has been successful, it has been used repeatedly with relatively minor modifications, and consequently is seen throughout biology. Specificity comes from using precise combinations of signaling molecules to achieve different ends in different cell types. Among the signaling systems that fit this description include the receptor and nonreceptor tyrosine kinases, G protein-coupled receptors, cell attachment receptors, guanylate cyclases, ligand-gated ion channels, the second messenger dependent and independent protein kinases and phosphatases, and transcription factors. The consequence of this conservation of molecular mechanisms is that many basic regulatory systems, which cannot be dissected free of the complexity of mammals, can be understood in relatively simple, manipulable experimental systems. With information from simple systems and the tools of genetics and molecular biology, complex problems such as human physiology and disease may be understood and eventually controlled.

Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle.

It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.