P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.     

Distribution of protein phosphatases type 1 and 2A in RINm5F cells

In the insulin producing cell line RINm5F distribution of serine/threonine specific protein phosphatases type 1 (PP1) and 2A (PP2A) was studied. Using different agents which inhibit or stimulate PP1 and PP2A we found that in membrane and nuclear fractions phosphatase activity was inhibited by okadaic acid (OA), protamine, heparin, and inhibitor-2 in a concentration-dependent manner. C2-ceramide had no effect. In the cytosolic fraction the inhibitory effect of okadaic acid was tenfold higher. Protamine stimulated phosphatase activity at low concentrations and became inhibitory at higher concentrations. Inhibitor-2 and heparin caused a decrease in phosphatase activity whereas C2-ceramide led to a slight activation. The data suggest that in membrane and nuclear fractions of RINmSF cells predominantly PP1 is present, whereas in the cytosol PP1 as well as PP2A can be detected.     

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.     

Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor

We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.     

Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A)

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.     

Regulation of N-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.     

An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primary rat hepatocyte cultures

We have previously demonstrated that specific activation of a cAMP-dependent protein kinase A (PKA) pathway resulted in complete repression of phenobarbital (PB)-inducible CYP gene expression in primary rat hepatocyte cultures. In the current investigation, we examined the role of protein phosphatase pathways as potential co-regulators of this repressive response. Primary rat hepatocytes were treated with increasing concentrations (0.1-25 nM) of okadaic acid, a potent inhibitor of serine/threonine-specific protein phosphatases PP1 and PP2A. PB induction responses were assessed by use of specific hybridization probes to CYP2B1 and CYP2B2 mRNAs. Okadaic acid completely inhibited the PB induction process in a concentration-dependent manner (IC50, approximately 1.5-2 nM). Similar repression was obtained with low concentrations of other highly specific phosphatase inhibitors, tautomycin and calyculin A. In contrast, exposure of hepatocytes to 1-nor-okadaone or okadaol, negative analogs of okadaic acid largely devoid of phosphatase inhibitory activity, was without effect on the PB induction process. At similar concentrations, okadaic acid produced only comparatively weak modulation of the beta-naphthoflavone-inducible CYP1A1 gene expression pathway. In additional experiments, hepatocytes were treated with suboptimal concentrations of PKA activators together with phosphatase inhibitors. Okadaic acid markedly potentiated the repressive effects of dibutyryl-cAMP on the PB induction process. Together, these results indicate that both PKA and protein phosphatase (PP1 and/or PP2A) pathways exert potent and complementary control of the intracellular processes modulating the signaling of PB in cultured primary rat hepatocytes.     

Protein phosphatases limit tumor motility

Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion.     

Relationships of subunits of type-1 serine/threonine protein phosphatase to morphology and aggregation of B cells

To elucidate the roles of serine/threonine protein phosphatases PP1 and PP2A in the morphological changes of B-lymphocytes during development and in immune responses, we investigated alterations of protein levels of catalytic subunits of PP1 and PP2A and regulatory subunits of PP1 including M130/M133, inhibitor-1 (I-1) and inhibitor-2 (I-2) in B-cell lines at different maturational stages and during their aggregation induced by phorbol myristate acetate (PMA). The protein levels of PP1delta and/or M130/M133 were significantly lower in B-cell lines without pseudopods, WEHI-231, BAL-17, Daudi, and CESS, than in those with pseudopods, Bcl.1, A20, M12, and SKW6.4, whereas the amounts of PP1alpha and PP2A were similar among them. During aggregation of A20 and CESS cells induced by PMA, an activator of PKC, the amount of PP1delta was progressively decreased, and this decrease was blocked by H7, an inhibitor of PKC. The amount of PP1alpha was constant under these conditions. Okadaic acid, an inhibitor of PP1 and PP2A, also induced aggregation of A20 cells at concentrations sufficient to inhibit PP1, but not at lower concentrations that inhibit PP2A alone. These results suggest that myosin light chain phosphatase composed of PP1delta and M130/M133 is involved in the maintenance and regulation of cytoskeletal structures in B-lymphocytes.     

Serine-70 is one of the critical sites for drug-induced Bcl2 phosphorylation in cancer cells

Taxoids and other microtubule-damaging drugs are known to induce Bcl2 phosphorylation at the G2-M phase of the cell cycle, with concomitant apoptosis in malignant cells derived from a variety of human malignancies, including leukemia, lymphoma, and breast and prostate cancer. We have investigated the ability of another antineoplastic drug, dolastatin 10, in inducing Bcl2 phosphorylation and apoptosis. We also investigated the effects of a phosphatase inhibitor okadaic acid in the regulation of Bcl2 phosphorylation, cell cycle arrest, and programmed cell death. Moreover, site-directed mutagenesis studies were performed to determine the specific serine residue(s) responsible for drug-induced Bcl2 phosphorylation. Our results indicate that these antimicrotubule agents or okadaic acid can induce posttranslational modification (phosphorylation) of Bcl2 protein at multiple serine residues. Interestingly, mutation of a serine residue at position 70 to alanine can significantly decrease drug-induced posttranslational modification (phosphorylation) of Bcl2 protein. Apparently, Ser70 seems to be a critical site for drug-induced posttranslational modification (phosphorylation) of the Bcl2 protein.     

Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits. Yet, the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.     

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.     

Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser70) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). In addition, an intact Ser70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A.     

Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein kinase pathway, inducing small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulosa cells

This study investigates the possibility that FSH activates the p38 mitogen-activated protein kinase (MAPK) pathway in immature granulosa cells (GC). FSH induced the phosphorylation (activation) of p38 MAPK as evaluated by immunoprecipitation and by phosphorylation-specific immunoblotting. FSH-induced phosphorylation of p38 MAPK was blocked by pretreatment with the protein kinase A (PKA) inhibitor H89 and mimicked by the cAMP generating agonist forskolin, indicating that FSH-induced cAMP production and PKA activation are necessary and sufficient for the activation of p38 MAPK in GC. The small heat shock protein HSP-27 comprises a downstream phosphorylation target for the p38 MAPK pathway. FSH-induced phosphorylation of HSP-27 was blocked by pretreatment with the p38 MAPK inhibitor SB 203580, indicating that p38 MAPK activation is necessary for FSH-induced HSP-27 phosphorylation. FSH-induced GC rounding/aggregation was blocked by pretreatment with SB 203580 indicating that p38 MAPK activation is necessary for FSH-induced GC cell shape change. The results of these experiments show that the p38 MAPK pathway is activated in GC in response to FSH in a cAMP/PKA-dependent manner, and that p38 MAPK activity is required for FSH-induced HSP-27 phosphorylation as well as rounding/aggregation in GC.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.