Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase Fa/GSK-3α in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C

The signal transduction mechanism of protein kinase FA /GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa /GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1–10 μM calphostin C (IC₅₀, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa /GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa /GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC₅₀, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa /GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa /GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA /GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.     

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK-3α in A431 cells

The signal transducrion mechanism of protein kinase F_A/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F_A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to ～60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37^oC for 30 min, as demonstrated by metabolic ³²P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase F_A/GSK-3α was found to be rephosphorylated on tyrosine residue and reactivated to ～130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase F_A/GSK-3α can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.     

Heat stress induces tyrosine phosphorylation/activation of kinase Fa/GSK-3α (a human carcinoma dedifferentiation modulator) in A431 cells

Exposure of A431 cells to a rapid temperature increase from 37° to 46°C could induce an increased expression (∼200% of control) and tyrosine phosphorylation/activation (∼300% of control) of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) in a time-dependent manner, as demonstrated by an anti-kinase FA/GSK-3α immunoprecipitate kinase assay and by immunoblotting analysis with anti-kinase FA/GSK-3α and anti-phosphotyrosine antibodies. The heat induction on the increased expression of kinase FA/GSK-3α could be blocked by actinomycin D but not by genistein. In contrast, the heat induction on tyrosine phosphorylation/activation of kinase FA/GSK-3α could be blocked by genistein or protein tyrosine phosphatase, indicating that heat stress induces a dual control mechanism, namely, protein expression and subsequent tyrosine phosphorylation to cause cellular activation of kinase FA/GSK-3α. Taken together, the results provide initial evidence that kinase FA/GSK-3α represents a newly described heat stress–inducible protein subjected to tyrosine phosphorylation/activation, representing a new mode of signal transduction for the regulation of this human carcinoma dedifferentiation modulator and a new mode of heat induction on cascade activation of a protein kinase. J. Cell. Biochem. 66:16–26, 1997. © 1997 Wiley-Liss, Inc.     

The Naturally Occurring PKC Inhibitor Sphingosine and Tumor Promoter Phorbol Ester Potentially Induce Tyrosine Phosphorylation/Activation of Oncogenic Proline-Directed Protein Kinase FA/GSK-3α in a Common Signalling Pathway

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase F_A/glycogen synthase kinase-3α (kinase F_A/GSK-3α) could be decreased to ~25% of control. Conversely, when treated with 1 μM TPA for 24 hr, the activity could be reversibly increased to ~200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 μM could also induce activation of kinase F_A/GSK-3α to ~200% of control within 60 min. Further, when cells were chronically treated with 1 μM TPA for 24 hr and then with 27 μM sphingosine for 60 min, the activity of kinase F_A/GSK-3α could only be increased to ~200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase F_A/GSK-3α activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase F_A/GSK-3α. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKC∈ in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.     

Dysfunction of protein kinase FA/GSK-3α in lymphocytes of patients with schizophrenic disorder

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP. Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA /GSK-3α immunoblotting and kinase activity analysis in an anti-kinase FA /GSK-3α immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA /GSK-3α in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA /GSK-3α activity in the high levels of 14.8 ± 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA /GSK-3α activity in the low levels of 2.8 ± 1.6 units/mg, indicating that the different levels of kinase FA /GSK-3α activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide intial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA /GSK-3α, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes. © 1995 Wiley-Liss, Inc.     

The Naturally Occurring PKC Inhibitor Sphingosine and Tumor Promoter Phorbol Ester Potentially Induce Tyrosine Phosphorylation/Activation of Oncogenic Proline-Directed Protein Kinase FA/GSK-3α in a Common Signalling Pathway

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) could be decreased to ~25% of control. Conversely, when treated with 1 M TPA for 24 hr, the activity could be reversibly increased to ~200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 M could also induce activation of kinase F_A/GSK-3 to ~200% of control within 60 min. Further, when cells were chronically treated with 1 M TPA for 24 hr and then with 27 M sphingosine for 60 min, the activity of kinase F_A/GSK-3 could only be increased to ~200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase F_A/GSK-3 activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3 in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase F_A/GSK-3. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKC in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.     

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA /GSK-3α in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 μM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA /GSK-3α was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA /GSK-3α immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA /GSK-3α can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA /GSK-3α. J. Cell. Physiol. 171:95–103, 1997. © 1997 Wiley-Liss, Inc.     

Tyrosin dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3α by genistein in A431 cells

Modulation of protein Kinase _F/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F _A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in concentration dependent manner by 50-400 μM genistein( a Specific tyrosine kinase inhibitor), as demonstrated by metobolic ³²p-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and byimmunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Taken together, the results provide evidence that Kinase F_A/GSK-3α may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can by tyrosine-dephosphorylated and nactivated by extracellular stimulus and that tyrosine kinase(s) and /or tyrosine phosphatase(s) may play a role in the modulation of kinse F_A/GSK-3α activity in cells.     

佛波酯促进NIH3T3细胞的铺展和聚焦粘附激酶的酪氨酸磷酸化

100nmol/L佛波酯(12-O-tetradecanoylphobol-13-acetate,TPA)能明显促进NIH3T3细胞在纤连蛋白(Fn)上的铺展,该作用能分别被酪氨酸激酶(tyrosinekinase,TK)抑制剂4′,5,7-三羟基异黄酮(genistein)和蛋白激酶C(proteinkinaseC,PKC)抑制剂calphostinC和神经鞘氨醇(sphingosine)所抑制.TPA作用于结合到Fn上的NIH3T3细胞,使其聚焦粘附激酶(focaladhe-sionkinase,FAK)的酪氨酸磷酸化程度较未处理细胞升高,于30min时达对照的204.0%,并存在浓度依赖性;该变化分别被上述抑制剂所拮抗;未经TPA处理的NIH3T3细胞和纤连蛋白结合诱导的FAK酪氨酸磷酸化亦分别被上述抑制剂所抑制.细胞松弛素D则无论TPA作用与否,都能完全阻断NIH3T3细胞的铺展和FAK的酪氨酸磷酸化.以上结果提示,TPA促进NIH3T3细胞在Fn上铺展的信号转导机制,与PKC的激活有关,进一步则可能通过影响FAK的酪氨酸磷酸化来实现,同时需要细胞骨架的参与;NIH3T3细胞和Fn结合并诱导FAK酪氨酸磷酸化的过程亦依赖于PKC和完整的细胞骨架.     

12‐O‐tetradecanoylphorbol‐13‐acetate‐induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF‐κB‐dependent MMP‐9 expression

An increase in MMP‐9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was detected in glioblastoma cells GBM8401. TPA‐induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) by TPA was identified, and TPA‐induced migration and MMP‐9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF‐κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF‐κB‐directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP‐9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA‐induced MMP‐9 activation and migration. Additionally, suppression of TPA‐induced PKCα/ERK/NK‐κB activation, migration, and MMP‐9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′‐tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′‐tetrahydroxyflavone), and wogonin (Wog; 5,7‐dihydroxy‐8‐methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP‐9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF‐κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley‐Liss, Inc.     

Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C.

The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PKC) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was 100-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (1 and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC.     

Phosphatidylinositol 3-kinase/Akt plays a role in sphingosine 1-phosphate-stimulated HSP27 induction in osteoblasts

We previously reported that p38 mitogen-activated protein (MAP) kinase plays a part in sphingosine 1-phosphate-stimulated heat shock protein 27 (HSP27) induction in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the induction of HSP27 in these cells. Sphingosine 1-phosphate time dependently induced the phosphorylation of Akt. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduced the HSP27 induction stimulated by sphingosine 1-phosphate. The sphingosine 1-phosphate-induced phosphorylation of GSK-3beta was suppressed by Akt inhibitor. The sphingosine 1-phosphate-induced HSP27 levels were attenuated by LY294002 or wortmannin, PI3K inhibitors. Furthermore, LY294002 or Akt inhibitor did not affect the sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase and SB203580, a p38 MAP kinase inhibitor, had little effect on the phosphorylation of Akt. These results suggest that PI3K/Akt plays a part in the sphingosine 1-phosphate-stimulated induction of HSP27, maybe independently of p38 MAP kinase, in osteoblasts.     

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f(2alpha)-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) activates both phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells and then induces the activation of protein kinase C (PKC). In this study, we investigated the effect of PGF(2alpha) on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein, in these cells. PGF(2alpha) significantly induced the accumulation of HSP27 dose-dependently within the range of 10 nM to 10 microM. PGF(2alpha) stimulated the increase in the levels of mRNA for HSP27. A total of 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, induced the accumulation of HSP27. The stimulative effect of PGF(2alpha) was reduced in the PKC down-regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF(2alpha)-induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF(2alpha) was reduced by U-73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF(2alpha) and TPA stimulated p42/p44 mitogen-activated protein (MAP) kinase. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, suppressed the induction of HSP27 stimulated by PGF(2alpha) or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF(2alpha). These results indicate that PGF(2alpha) stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts.     

C-kinase manipulations disrupt activity-driven retinotopic sharpening in regenerating goldfish retinotectal projection

Regenerating optic axons initially branch over a wide area in tectum to form a crude retinotopic map. The map is sharpened, and retinotopically appropriate synapses are stabilized via NMDA receptors that detect, via summation of EPSPs, the coincident activity of neighboring ganglion cells that make synapses onto common tectal cells. Sharpening shares a number of properties with long-term potentiation (LTP) in hippocampus. This study tested whether protein kinase C (PKC) activation is necessary for sharpening as it is for LTP. Intracular (IO) or intracranial (IC) injections of kinase inhibitors or activators were made every other day from 19 to 37 days postcrush (sensitive period), and the projections formed were later recorded. Retinotopic sharpening was prevented by IC injection of the following agents: (1) general kinase inhibitors sphingosine and H7 (100-200 μM in fluid above brain), (2) active but not inactive phorbols (TPA, 1 μM), and (3) calphostin C (1 μM), a specific and irreversible PKC inhibitor. The mature projection on the opposite tectum, however, when examined was not unsharpened. Lack of sharpening was reflected in multiunit fields at each tectal point that averaged 27°–30° versus 11° in Ringers and inactive phorbol control regenerates. Intraocular injections of either TPA (1 μM), or calphostin C (1 μM) also prevented sharpening (26° and 32° multiunit fields), suggesting action on PKC axonally transported to the presynaptic terminals. Calphostin C had no noticeable effect on the firing patterns of retinal ganglion cells. The endogenous activator of PKC, arachidonic acid (AA), disrupted sharpening at 20 μM or higher (IC injection, 32° multiunit fields), while a control fatty acid, elaidic acid, had no effect. Although AA at 5 μM showed no effect, and diacylglycerol at 5 μM exhibited only small effects, together they produced a large synergistic effect (32° multiunit fields). Such synergy mirrors the synergy in the activation of several isoforms of PKC. Actual concentrations in the extradural fluid around the brain were assayed via injections of ³H-AA. Levels fell about sixfold after a day and by an additional fivefold the second day before the next injection. The results confirm that activity-driven retinotopic sharpening is very sensitive to manipulations of kinases, especially PKC. © 1994 John Wiley & Sons, Inc.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.     

Protein kinase C-induced phosphorylation modulates the Na(+)-ATPase activity from proximal tubules

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P<0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.