Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5-10 microM) resulted in a dose-dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on attachment to fibronectin. Phorbol 12-myristate 13-acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.     

Retinoic acid modulates attachment of mouse fibroblasts to laminin substrates

The effect of retinoic acid treatment on cell attachment to plastic substrates precoated with fibronectin, gelatin, laminin, and type IV collagen was investigated. Both retinoic acid-treated and control cells attached efficiently to fibronectin or gelatin substrates without any significant difference. In contrast, retinoic acid-treated cells attached to laminin or type IV collagen substrates, while control cells showed little or no attachment. The minimal effective concentration of retinoic acid for pretreatment to yield a significant increase in the attachment assay was higher than 10(-8) M. The attachment of retinoic acid-treated cells to laminin substrates reached a maximum at 60 min, while that to type IV collagen substrates had a time lag and did not reach a maximum by 60 min. The effect of retinoic acid treatment reached a maximum at 2 days and was partly reversible. These results suggest that retinoic acid may increase NIH/3T3 cell adhesion through an effect on laminin receptors. Other mouse fibroblast lines, 3T3-Swiss, 3T6-Swiss, Balb/3T3, and Balb/3T12-3 (spontaneously transformed Balb/3T3), responded to retinoic acid treatment in a manner similar to that of NIH/3T3 cells. However, the virus-transformed Balb/3T3 lines, SV-T2 and M-MSV, showed significant attachment to laminin substrates without retinoic acid treatment, and retinoic acid did not affect or slightly decreased the cell attachment to laminin substrates.     

Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines

The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the α 6/β1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexaminde, can be blocked by monoclonal antibodies (Mabs) to the β1 and α 5 subunits of the β1 family of integrins, and is not associated with increased expression of the α 5 or β1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48–72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the β1 and α 6 subunits, and is associated with increased expression of the α 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4–120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in α /β1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines. © 1993 Wiley-Liss, Inc.     

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.     

Lovastatin and phospholipase Cgamma regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen

We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.     

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.     

The protein phosphatase inhibitor, okadaic acid, potentiates the stimulatory effect of phorbol ester on phosphatidylcholine synthesis, but not on phospholipid hydrolysis, in fibroblasts.

The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester-induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2 microM) was found to enhance the stimulatory effects of lower concentrations (2.5-25 nM) of phorbol 12-myristate 13-acetate (PMA) on PtdCho synthesis, but not on PtdCho hydrolysis, after treatments for 30-60 min. These data support a view that in fibroblasts PMA stimulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation-dependent mechanism.     

1,2-Diacylglycerol does not mediate the stimulatory effect of phorbol 12-myristate 13-acetate on phosphatidylcholine synthesis in NIH 3T3 fibroblasts.

In HeLa cells, increased 1,2-diacylglycerol (1,2-DAG) has been suggested to mediate the stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on phosphatidylcholine (PtdCho) biosynthesis (A. K. Utal, H. Jamil, and D. E. Vance, 1991, J. Biol. Chem. 266, 24,084-24,091). The aim of this study was to examine if 1,2-DAG might have a similar mediatory role in NIH 3T3 fibroblasts. In these cells, PMA-induced hydrolysis of PtdCho and the formation of secondary product 1,2-DAG was inhibited by exposing the cells to either 300 mM ethanol for 15 min (less than 80% inhibition) or 43 degrees C for 60 min (less than 50% inhibition). In contrast, neither ethanol nor heat-treatment caused significant inhibition of PMA-stimulated PtdCho synthesis. These data indicate that in NIH 3T3 fibroblasts, 1,2-DAG is not a mediator of the stimulatory action of PMA on PtdCho synthesis.     

Alterations in cell surface carbohydrate composition of a human colon carcinoma cell line affect adhesion to extracellular matrix components.

The adhesion of HT29 human colon adenocarcinoma cells to different extracellular matrix components was studied. While treatment of the cells with sialidase had no detectable effect on binding to laminin and fibronectin, attachment to collagen IV was decreased. However, additional removal of beta-(1-4)-bound galactose led to significantly reduced binding to all of the substrates, including fibronectin and laminin. Tunicamycin treatment, monitored by lectin-induced aggregation, drastically diminished cell adhesion to laminin and fibronectin, whereas cell binding to collagen IV was not affected. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion to collagen IV. The results show that a serine-containing RGD-related peptide GRGDSP has virtually no effect on colon carcinoma cell adhesion to type IV collagen. In contrast, when serine was substituted for threonine (GRGDTP) adhesion to collagen IV was strongly inhibited. After incubation of sialidase-treated cells with the threonine-containing peptide adhesion was almost totally blocked. These results demonstrate the existence of both RGD-dependent and carbohydrate-based mechanisms for metastatic human HT29 cell binding to collagen IV.     

3T3 cell surface galactosyltransferase is a calcium-dependent adhesion molecule for collagen type IV.

Cell surface galactosyltransferase (GalTase) has been previously shown to mediate cell spreading or migration on laminin matrices. This work demonstrates that 3T3 cell surface GalTase also mediates cell attachment to collagen type IV. Attachment to collagen type IV was blocked by perturbations of GalTase or substrate pregalactosylation on cells possessing only calcium-dependent mechanisms of adhesion. Cells with both calcium-dependent and calcium-independent systems were not affected by GalTase perturbation. Collagen type IV was shown to possess GalTase substrates since matrices could be galactosylated by both soluble enzyme and 3T3 cells.     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT)

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.     

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type XVII collagen (BP180) can function as a cell−matrix adhesion molecule via binding to laminin 332

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knock-down of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix.     

Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex

We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.