- Research article
- Open Access
Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α1A adrenergic receptor
© Parmentier et al; licensee BioMed Central Ltd. 2004
Received: 07 October 2003
Accepted: 21 January 2004
Published: 21 January 2004
Phenylephrine (PHE), an α1 adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.
PHE stimulated a PLD activity as demonstrated by phosphatidylethanol production. PHE increased PKCζ translocation to the particulate cell fraction in parallel with a time-dependent decrease in its activity. PKCζ activity was reduced at 2 and 5 min and returned to a sub-basal level within 10–15 min. Ectopic expression of kinase-dead PKCζ, but not constitutively active PKCζ, potentiated PLD activation elicited by PHE. A cell-permeable pseudosubstrate inhibitor of PKCζ reduced basal PKCζ activity and abolished PHE-induced PLD activation.
α1A adrenergic receptor stimulation promotes the activation of a PLD activity by a mechanism dependent on PKCζ; Our data also suggest that catalytic activation of PKCζ is not required for PLD stimulation.
Phospholipase D (PLD) is widely distributed in mammalian cells and has been shown to be involved in signal transduction, protein trafficking, cell proliferation, differentiation and apoptosis [1–3]. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. Activation of PLD by various agents has been shown to involve small G-proteins of the Arf and Rho families, protein kinase C (PKC) and phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) [1–3]. Two PLD isoforms have been cloned in humans and rats. PLD1 exhibits a low basal activity and is activated by Arf, RhoA and PKC [4, 5]. PLD2 has a high basal activity, requires PtdIns(4,5)P2, and is not or is less responsive to Arf, Rho or PKC than PLD1 [6, 7].
Stimulation of α1 adrenergic receptors (AR) increases PLD activity in rat tail artery  and MDCK cells . In rat-1 fibroblasts expressing different subtypes of α1 AR, α1A AR is more effectively coupled to PLD activation than other α1 AR subtypes [10, 11].
The involvement of PKC in PLD regulation has been documented both in vivo and in vitro [1–3]. PKC isoforms are classified on the basis of their protein sequences and biochemical properties . The classical PKC isoforms (α, β1,β2 and γ) are activated by phosphatidylserine and diacylglycerol (DAG) or phorbol esters in a calcium-dependent manner. The novel PKC isoforms (δ, ε, Η and θ) are activated by DAG or phorbol esters in the presence of phosphatidylserine and in the absence of calcium. Classical and novel PKCs play a critical role in cell proliferation, differentiation, tumorigenesis, and apoptosis and have a multitude of cellular substrates with broadly overlapping specificity [12, 13]. The atypical PKC isoforms (ι/λ and ζ) are both calcium- and DAG-independent . PKCζ is a critical mediator of mitogenic signaling in many cell types [13–16]. The activation of PI3-kinase by growth factors induces a moderate activation of PKCζ that is mediated by phosphorylation at its T-loop site by PDK1 followed by a subsequent autophosphorylation [17, 18]. The activity of PKCζ is reversibly regulated by an autoinhibitory pseudosubstrate region in the regulatory domain, which blocks the active site of the enzyme in the absence of activators, a feature common to all PKCs . In addition, the PKCζ pseudosubstrate is able to interact with tubulin and p62/ZIP protein [20, 21]. PKCζ is activated by nonselective binding of acidic lipids such as polyphosphoinositides and phosphatidic acid, unsaturated fatty acids such as arachidonic acid , and acidic proteins such as 14-3-3 proteins . In rat-1 fibroblasts, PKCζ mediates the activation of ERK and the increase in mitogenesis elicited by PDGF . However, in rat-1 fibroblasts expressing the α1A AR subtype, norepinephrine does not activate ERK .
Classical PKC subtypes have been implicated in PLD activation in vitro or in cells overexpressing classical PKCs [3, 24]. However, there are reports indicating receptor-mediated PLD activation that is independent of classical PKCs [9, 25]. PLD activation by classical PKCs in vitro does not involve a phosphorylation mechanism . It is currently unclear if the non-catalytic mechanism by which PKCα and β activate PLD1 in vitro accounts for PKC-dependent increases in PLD activity in intact cells [1–3]. We have previously reported that α1A adrenergic stimulation of PLD in rat-1 fibroblasts is independent of classical or novel PKCs . Three recent articles have placed activation of atypical PKCs downstream of PLD, presumably through phosphatidic acid generation [27–29]. On the other hand, PKCζ mediates norepinephrine-induced PLD activation in rabbit vascular smooth muscle cells (VSMC) . The present study was conducted to investigate the relationship between PKCζ and PLD activation in response to PHE in rat-1 fibroblasts expressing the α1A AR subtype. Our study demonstrates that PHE stimulates a PLD activity in rat-1 fibroblasts by a mechanism dependent on the inactivation of PKCζ activity and suggests a role for the pseudosubstrate domain.
PHE and PMA stimulate PLD activity in rat-1 fibroblasts
PKCζ is inactivated in response to α1A-adrenergic stimulation
To gain more insight into the mechanism of PKCζ regulation by PHE, we measured PKCζ activity in immunoprecipitates from cells pretreated with myristoylated PKCζ peptide inhibitor (PSζ) . Myristoylated PSζ reduced basal PKCζ activity and prevented its further decrease by PHE (Fig. 5B), confirming that PKCζ is active in untreated cells arrested in serum-free medium. It should be noted that PHE produced a significantly greater decrease in PKCζ activity than myristoylated PSζ, alone or with PHE, pointing to a closely regulated mechanism of PHE-induced PKCζ inactivation. Together, these results show that PHE inhibits PKCζ and simultaneously activates PLD in rat-1 fibroblasts.
Effect of PKCζ on PLD activity
A PKCζ inhibitor blocks PHE-induced PLD activity
This study confirms that stimulation of α1A AR with PHE in rat-1 fibroblasts promotes activation of a PLD activity [10, 11, 25]. Moreover, it demonstrates that PHE selectively decreases PKCζ activity and that PLD activity is regulated by a mechanism involving PKCζ. Furthermore, the pseudosubstrate domain of PKCζ appears to play an important role in the regulation of PLD. Our previous study has ruled out the involvement of classical or novel PKC isoforms in α1A AR-stimulated PLD activation .
In rat-1 fibroblasts expressing the α1A AR subtype, PHE causes a transient Ca2+ increase, cAMP accumulation and activation of PKA, p38 mitogen-activated protein kinase, p70 S6 kinase and PLD activation [10, 11, 23, 34]. In contrast, PHE inhibits basal levels or agonist-induced activation of ERK, PI 3-kinase and Akt in these cells [11, 34]. In addition, PHE slightly decreases basal PtdInsP2 and PtdInsP3 levels . Despite these negative effects on proliferation (ERK) and survival (PI 3-kinase, Akt) pathways in rat-1 fibroblasts, α1A AR stimulation does not significantly promote apoptosis . It should be noted that in these cells, PLD activity is extremely low both in serum-deprived or serum-treated cells; PLD activation by PHE is not affected by the presence of serum and PHE decreases cell proliferation (Parmentier JH, Saeed AE and Malik KU, our unpublished observation). These observations, together with our finding that PHE-induced decrease in PKCζ activity is associated with an increase in PLD activity, support an anti-proliferative effect of α1A AR stimulation in rat-1 fibroblasts, involving PLD activation. In contrast, in VSMC expressing high levels of the PLD2 isoform [30, 32], norepinephrine-induced cell proliferation was dependent on PLD activation. In addition, norepinephrine selectively stimulates the PLD2 isoform in VSMC  through the catalytic activation of PKCζ , in contrast to the effect of PHE in rat-1 fibroblasts. The cell phenotype, VSMC vs. rat-1 fibroblasts, or the adrenergic receptor subtype, α1A AR in rat-1 vs. several α1 and α2 AR subtypes in VSMC, may be responsible for this difference.
To our knowledge, this is the first report demonstrating inhibition of PKCζ activity in response to receptor stimulation. However, treatment with PHE decreases the activity of many mitogenic indices in rat-1 fibroblasts, including DNA synthesis, ERK, phosphatidylinositol 3-kinase and Akt [11, 34]. A noteworthy fact is that there is elevated mitogenic signaling in rat-1 fibroblasts expressing α1A AR cultured in serum-free medium. The decrease in PKCζ activity elicited by PHE could result from a physical interaction between PKCζ and K10 keratin, causing sequestration of PKCζ within the cytoskeleton and preventing its intracellular translocation, thus impairing its activation, as reported by Paramio et al. , although translocation is often associated with activation for other PKC isoforms. In addition, a recent study shows that a small pool of PKCζ is constitutively active and bound to 14-3-3 zeta in the brain , a mechanism that could account for the persistent activation of PKCζ in rat-1 fibroblasts. PI 3-kinase, an upstream activator required for PKCζ activation, is also inhibited by PHE in rat-1 fibroblasts . Recently, it has been demonstrated that atypical PKC activation is dependent on PLD activity [27–29]. However, our data show that PLD activation with PHE was associated with a decrease in PKCζ activity in rat-1 fibroblasts.
Our data also shows that the decrease in PKCζ activity elicited by PHE is not matched by a decrease in the phosphorylation of Thr 410. PKCζ is maintained in an inactive state by direct binding of the N-terminal pseudosubstrate domain to the C-terminal catalytic domain . Phosphorylation of the activation loop at Thr 410 is necessary and sufficient to activate the kinase function of PKCζ after autophosphorylation of Thr 560 [36–38]. In VSMC, we observed a simultaneous increase in PKCζ phosphorylation and activity in response to norepinephrine . However, at 5 min of stimulation, PKCζ phosphorylation continued to increase whereas PKCζ activity was already decreasing . Therefore, although phosphorylation at Thr 410 is a prerequisite for PKCζ activation as shown by different studies [36–38], it is not clear if dephosphorylation of Thr 410 is a prerequisite for its inactivation. It has been recently reported that in fact phosphorylation of Thr 410 and subsequent autophosphorylation of Thr 560 targets PKCζ towards proteosomal degradation . Moreover, proteins that bind to PKCζ may directly inhibit its activity. For example, PAR-4, product of a gene induced during apoptosis, inhibits atypical PKCζ and PKCλ/ι activity through direct protein-protein interaction . This mechanism of inhibition of PKCζ activity may not require dephosphorylation of the enzyme. Thus, phosphorylation at Thr 410 seems to be required for PKCζ activation whereas inhibition of PKCζ activity seems to be independent of Thr 410 dephosphorylation and may involve other proteins. Since PKCζ immunoprecipitation and assay were carried out in non-denaturing conditions, it is likely that the interaction of PKCζ with other proteins, such as with an endogenous inhibitor, is conserved during the assay.
The activation of PLD elicited by PHE may be independent of PKCζ catalytic activity since constitutively active T410E PKCζ did not alter the extent of PHE-induced PLD activation. It is widely accepted that PKCζ activates downstream targets through a phosphorylation-dependent mechanism. However, it has been reported that atypical PKCζ may also directly stimulate MEK5/ERK5 pathway through its N-terminal regulatory domain (containing the pseudosubstrate site), independent of its catalytic activity . Therefore, PKCζ may stimulate downstream targets independent of its activity and phosphorylation state. Moreover, the actual proposed mechanism of PLD1 activation by the classical isoform PKCα is independent of the catalytic activity of PKC . In contrast, the fact that catalytically inactive PKCζ potentiated PLD activity and PKCζ activity was decreased after PHE stimulation indicate that an initial decrease in PKCζ activity may be required for a non-catalytic PKCζ-dependent activation of PLD activity. On the other hand, the decrease in PKCζ activity could be a consequence and not a prerequisite for this potential mechanism of action.
Regulation of PLD activity by PKCζ may involve the pseudosubstrate domain or a regulatory domain of PKCζ. PSζ is utilized to inhibit PKCζ activity [14, 30]. However, we show that PHE is a better inhibitor than PSζ and PSζ further blocks the decrease in PKCζ activity elicited by PHE. These data and the lack of decrease in PKCζ phosphorylation suggest that other proteins activated by PHE may be involved in reducing PKCζ activity. In addition, PSζ blocked PHE-induced PLD activation. Therefore, PSζ may alter PKCζ function(s) through two different actions. First, it inhibits kinase activity through binding to the catalytic site, which mimics the endogenous pseudosubstrate domain [14, 33]. Second, it may act a competitive inhibitor of the interaction of the regulatory N-terminal domains (PB1, PS, C1) [42, 43] of PKCζ with other effectors, such as PAR, MEK5, p62/ZIP, tubulin or other proteins [20, 21, 40, 41, 44, 45]. PKCζ directly stimulates the MEK5/ERK5 pathway through its N-terminal regulatory domain independently of its catalytic activity . PSζ may competitively inhibit the interaction of the regulatory domain with a protein containing an aPKC interaction domain, such as MEK-5 or the scaffold protein p62, in addition to its effect on catalytic activity. This mechanism of action would explain the effect of PSζ on PLD activity and PKCζ activity. It should be noted that a PKCα pseudosubstrate inhibitor did not alter PHE-induced PLD activity (Fig. 8), underscoring the selectivity of myristoylated PSζ.
An alternative hypothesis involves a PKCζ-mediated PLD phosphorylation that would keep PLD inactive when PKCζ is active. However, our data do not support this, since kinase inactive PKCζ or myristoylated PSζ did not stimulate PLD activity in the absence of PHE stimulation. Moreover, PLD phosphorylation is probably not involved in its mechanism of activation .
PKCα, the only classical isoform found in rat-1 fibroblasts expressing α1A AR, was not activated, as previously shown in these cells . A previous study by Taguchi et al.  showed an increase in PKCα translocation in response to PHE in the same cell model. The main difference between the two studies resides in the PHE concentration, 2 μM PHE in our work vs. 100 μM PHE , indicating that PHE is able to significantly stimulate PKCα translocation at concentration higher than 2 μM.
Our data indicate that a non-constitutively active PLD isoform expressed in rat-1 fibroblasts is regulated by a PKCζ-dependent mechanism following α1A AR stimulation by PHE. The exact mechanism of PLD activation by PKCζ is still unknown and may be independent of the catalytic activity. Our data show the possible regulation of PLD by a mechanism involving the pseudosubstrate or the regulatory domain of PKCζ. Further characterization of the mechanism of PLD activation and the possible interaction of PLD isoforms with PKCζ should provide important information about its regulation and its functional significance in rat-1 fibroblasts.
Phenylephrine was obtained from Sigma (St. Louis, MO); phorbol-12-myristate-13-acetate (PMA) from Calbiochem (San Diego, CA); myristoylated PKCζ and PKCα/β (20–28) peptide inhibitors from Biomol (Plymouth Meeting, PA); [3H]oleic acid (50 Ci/mmol) from American Radiolabeled Chemicals (St. Louis, MO); [γ-32P]ATP (3000 Ci/mmol) from Amersham (Arlington Heights, IL). Antibodies to PKCα, PKCζ and p38 were from Santa-Cruz Biotechnology (Santa Cruz, CA); phospho-PKCζ/λ and phospho-p38 antibodies were from Cell Signaling Technology (Beverly, MA).
Rat-1 fibroblasts were stably transfected with bovine α1A AR (a kind gift from Drs. L.F. Allen and R.J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC). Cells were maintained under 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 50 units of penicillin, 50 μg of streptomycin per ml, and 10% fetal bovine serum (FBS). Selection and maintenance of stably transfected cells were carried out with 400 μg/ml G418 (Invitrogen, San Diego, CA).
Rat-1 fibroblasts were transiently transfected with pCMV-FLAG-wt-PKCζ, pCMV-FLAG-T410A-PKCζ (kinase inactive), pCMV-FLAG-T410E-PKCζ (constitutively active) vectors (gift from Dr. A. Toker, BBRI, Boston, MA and R. Farese, USF, Tampa, FL) using Lipofectamine PLUS (Invitrogen). Efficiency of transfection was determined by Western blot analysis using an anti-FLAG antibody (Sigma). Transfection of rat-1 fibroblasts was performed for 2 days before assays with cells washed 8 hours after transfection to avoid lipofectamine-induced cytotoxicity.
Phospholipase D assay
PLD activity was quantitated by the formation of phosphatidylethanol according to a method previously described for rat-1 fibroblast . Briefly, serum-starved rat-1 fibroblasts in 6-well plates were labeled overnight with [3H]oleic acid (1 μCi/ml) in DMEM. Cells were then incubated with inhibitors and exposed to PHE (2 μM) for 15 minutes in the presence of 200 mM ethanol. Rat-1 fibroblasts were scraped into 2 ml ice-cold methanol and HCl solution, and 1 ml chloroform was added. Lipids were separated by chloroform extraction. A 40 μl aliquot was removed from the chloroform phase to determine the content of radioactivity in the total lipid fraction. The chloroform phase (0.8 ml) was evaporated under nitrogen and redissolved in 50 μl chloroform/methanol (9:1) containing phosphatidylethanol standard. Samples were spotted onto a silica gel thin-layer chromatography plate, and lipids were separated with the solvent system of chloroform/acetone/methanol/HCl/water (50:20:12.5:10:7.5). Phosphatidylethanol was identified by the mobility of authentic standard visualized with iodine vapor. Lanes containing phosphatidylethanol were scraped, and radioactivity was measured by scintillation spectroscopy. Data were measured as the ratio of [3H]phosphatidylethanol to [3H]total lipids.
The activity of PKCζ was determined according to the method described [14, 24]. Rat-1 fibroblasts in 100 mm dish were washed with PBS and scraped in 1 ml RIPA buffer [50 mM Tris.Cl, pH 7.4; 150 mM NaCl, 1% IGEPAL, 1 mM EDTA] containing protease and phosphatase inhibitors [1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, 1 mM Na orthovanadate, 1 mg/ml p-nitrophenyl-phosphate]. Total cell lysates were incubated with rabbit polyclonal PKCζ antibody (Santa Cruz Biotechnology, CA) for three hours and the immunocomplex was captured with a 50% slurry of protein A agarose beads. PKCζ immunoprecipitates were washed twice with high salt [50 mM Tris.Cl, pH 7.5; 10 mM MgCl2; 0.5 M LiCl] and low salt [50 mM Tris.Cl, pH 7.5; 10 mM MgCl2] buffers, and incubated with a kinase buffer [50 mM Tris.Cl, pH 7.5; 10 mM MgCl2; 0.2 mM EGTA, 50 μM ATP] containing 50 μM ε-peptide and 3 μCi [γ-32P]ATP at 30°C. The reaction was stopped by the addition of 200 mM EDTA and the proteins were precipitated by the addition of 25% TCA. The solutions were centrifuged for one minute at 14,000 rpm, and supernatants spotted onto p81 phosphocellulose filters. Filters were washed with 1% (v/v) orthophosphoric acid and analyzed by Cerenkov counting. PKCζ activity was calculated from the amount of 32P incorporated into the ε-peptide.
Western blot Analysis
Total cell lysate was prepared in modified RIPA buffer containing protease and phosphatase inhibitors and protein concentration was determined by the Bradford method. Proteins in 50 μg of lysate were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with antibodies at dilutions recommended by the manufacturers (Cell Signaling Technology and Santa-Cruz Biotechnology). The blots were visualized with the ECL plus detection system (Amersham Pharmacia, Piscataway, NJ). For PKCζ phosphorylation at Thr 410, an indicator of PKCζ kinase activity, a phospho-PKCζ/λ antibody was used. Blots were also stripped off with a stripping buffer [100 mM β-mercaptoethanol; 62.5 mM Tris.Cl, pH 6.7, 2% SDS] for 30 min at 50°C, washed twice with TBST, and reprobed with nPKCζ or p38 antibody.
For experiments involving cell fractionation, cells cultured in 150 mm dishes (two per treatment) were scraped in fractionation buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM sodium orthovanadate, 10 μg/ml trypsin inhibitor), subjected to 25 passes in a Potter-type teflon-on-glass homogenizer with a loose fitting and centrifuged at 700 × g for 5 min. The supernatant was centrifuged at 100,000 × g for 30 min, and the cytosolic fraction was stored at -80°C. The particulate fraction was resuspended in 1% Triton-RIPA buffer, sonicated, and stored at -80°C. 50 μg aliquots of each fraction were analyzed by Western-blot.
The results are expressed as mean ± SE. The data were analyzed by one-way ANOVA. The unpaired Student's t-test was applied to determine the difference between two groups, and the Newman-Keuls' a posteriori test to determine the difference between multiple groups. A value of P = 0.05 was considered significant. PLD activity was expressed as either 1000 × [3H]phosphatidylethanol / [3H]total lipids, or as a fraction of the basal or as the fold increase over vehicle. The phosphoprotein and protein level were estimated by densitometric analysis of the Western blots and performed on the indicated number of blots using NIH Image software, and expressed as a fold increase or decrease (mean ± SE) of the control, arbitrarily chosen as 1.
Jean-Hugues Parmentier carried out PLD assays, fractionation and Western blot studies, and transfection experiments. Gautam K. Gandhi performed most of the PKCζ assay and some Western blot analysis. Monique T. Wiggins prepared the PKCζ constructs. Abdelwahab E. Saeed performed some PLD activity assays. Sylvain G. Bourgoin assisted in the design of the study and analysis of the data. Kafait U. Malik coordinated the study. All authors read and approved the final manuscript.
This research was supported by NIH-NHLBI grant 19134-29. JHP was supported by a grant of the American Heart Association (Southeast Affiliates). GKG was supported by UT Graduate Health Science grant. AES was supported by a NIH Minority Postdoctoral Fellowship, supplement to NIH-NHLBI grant 19134-29. We thank Anne Estes for her technical assistance and Dr. Lauren Cagen for editorial comments. We gratefully acknowledge Dr. Alex Toker (BBRI, Boston, MA) and Dr. Robert Farese (University of South Florida, Tampa, FL) for generously supplying us with the PKCζ plasmids.
- Frohman MA, Morris AJ: Phospholipase D structure and regulation. Chem Phys Lipids. 1999, 98: 127-140. 10.1016/S0009-3084(99)00025-0.View ArticlePubMedGoogle Scholar
- Liscovitch M, Czarny M, Fiucci G, Tang X: Phospholipase D: molecular and cell biology of a novel gene family. Biochem J. 2000, 345: 401-415. 10.1042/0264-6021:3450401.PubMed CentralView ArticlePubMedGoogle Scholar
- Exton JH: Regulation of phospholipase D. FEBS Lett. 2002, 531: 58-61. 10.1016/S0014-5793(02)03405-1.View ArticlePubMedGoogle Scholar
- Hammond SM, Jenco JM, Nakashima S, Cadwallader K, Gu Q, Cook S, Nozawa Y, Prestwich GD, Frohman MA, Morris AJ: Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J Biol Chem. 1997, 272: 3860-3868. 10.1074/jbc.272.6.3860.View ArticlePubMedGoogle Scholar
- Park SK, Provost JJ, Bae CD, Ho WT, Exton JH: Cloning and characterization of phospholipase D from rat brain. J Biol Chem. 1997, 272: 29263-29271. 10.1074/jbc.272.46.29263.View ArticlePubMedGoogle Scholar
- Colley WC, Sung TC, Roll R, Jenco J, Hammond SM, Altshuller Y, Bar-Sagi D, Morris AJ, Frohman MA: Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr Biol. 1997, 7: 191-201.View ArticlePubMedGoogle Scholar
- Kodaki T, Yamashita S: Cloning, expression, and characterization of a novel phospholipase D complementary DNA from rat brain. J Biol Chem. 1997, 272: 11408-11418. 10.1074/jbc.272.17.11408.View ArticlePubMedGoogle Scholar
- Labelle EF, Fulbright RM, Barsotti RJ, Gu H, Polyak E: Phospholipase D is activated by G protein and not by calcium ions in vascular smooth muscle. Am J Physiol. 1996, 270: H1031-H1037.PubMedGoogle Scholar
- Balboa MA, Insel PA: Stimulation of phospholipase D via alpha1-adrenergic receptors in Madin-Darby canine kidney cells is independent of PKC alpha and -epsilon activation. Mol Pharmacol. 1998, 53: 221-227.PubMedGoogle Scholar
- Ruan Y, Kan H, Parmentier JH, Fatima S, Allen LF, Malik KU: Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A. J Pharmacol Exp Ther. 1998, 284: 576-585.PubMedGoogle Scholar
- Taguchi K, Yang M, Goepel M, Michel MC: Comparison of human alpha1-adrenoceptor subtype coupling to protein kinase C activation and related signaling pathways. Naunyn-Schmiedeberg's Arch Pharmacol. 1998, 357: 100-110.View ArticleGoogle Scholar
- Mellor H, Parker PJ: The extended protein kinase C superfamily. Biochem J. 1998, 332: 281-292.PubMed CentralView ArticlePubMedGoogle Scholar
- Toker A: Signaling through protein kinase C. Front Biosci. 1998, 3: 1134-1147.Google Scholar
- Liao DF, Monia B, Dean N, Berk B: Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem. 1997, 272: 6146-6150. 10.1074/jbc.272.10.6146.View ArticlePubMedGoogle Scholar
- Van Dijk MC, Muriana JG, Van der Hoeven PCJ, De Widt J, Schaap D, Moolenar WH, Van Blitterswijk WJ: Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta. Biochem J. 1997, 323: 693-699.PubMed CentralView ArticlePubMedGoogle Scholar
- Sajan MP, Standaert ML, Bandyopadhyay G, Quon MJ, Burke TR, Farese RV: Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes. J Biol Chem. 1999, 274: 30495-30500. 10.1074/jbc.274.43.30495.View ArticlePubMedGoogle Scholar
- Balendran A, Biondi RM, Cheung PCF, Casamayor A, Deak M, Alessi DR: A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase C zeta (PKC zeta) and PKC-related kinase 2 by PDK1. J Biol Chem. 2000, 275: 20806-20813. 10.1074/jbc.M000421200.View ArticlePubMedGoogle Scholar
- Standaert ML, Bandyopadhyay G, Perez L, Price D, Galloway L, Poklepovic A, Sajan MP, Cenni V, Sirri A, Moscat J, Toker A, Farese RV: Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem. 1999, 274: 25308-25316. 10.1074/jbc.274.36.25308.View ArticlePubMedGoogle Scholar
- Dutil EM, Newton AC: Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol. J Biol Chem. 2000, 275: 10697-10701. 10.1074/jbc.275.14.10697.View ArticlePubMedGoogle Scholar
- Garcia-Rocha M, Avila J, Lozano J: The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain. Exp Cell Res. 1997, 230: 1-8. 10.1006/excr.1996.3364.View ArticlePubMedGoogle Scholar
- Puls A, Schmidt S, Grawe F, Stabel S: Interaction of protein kinase C zeta with ZIP, a novel protein kinase C-binding protein. Proc Natl Acad Sci USA. 1997, 94: 6191-6196. 10.1073/pnas.94.12.6191.PubMed CentralView ArticlePubMedGoogle Scholar
- Dai JG, Murakami K: Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain. J Neurochem. 2003, 84: 23-34. 10.1046/j.1471-4159.2003.01254.x.View ArticlePubMedGoogle Scholar
- Alexandrov A, Keffel S, Goepel M, Michel MC: Stimulation of alpha1A-adrenoceptors in Rat-1 cells inhibits extracellular signal-regulated kinase by activating p38 mitogen-activated protein kinase. Mol Pharmacol. 1998, 54: 755-760.PubMedGoogle Scholar
- Mukherjee JJ, Chung T, Ways DK, Kiss Z: Protein kinase C alpha is a major mediator of the stimulatory effect of phorbol ester on phospholipase D-mediated hydrolysis of phosphatidylethanolamine. J Biol Chem. 1996, 271: 28912-28917. 10.1074/jbc.271.46.28912.View ArticlePubMedGoogle Scholar
- Parmentier JH, Ahmed A, Ruan Y, Gandhi GK, Saeed AE, Malik KU: Calcium and protein kinase C (PKC)-related kinase mediate alpha 1A-adrenergic receptor-stimulated activation of phospholipase D in rat-1 cells, independent of PKC. J Pharmacol Exp Ther. 2002, 303: 1206-1215. 10.1124/jpet.102.041384.View ArticlePubMedGoogle Scholar
- Singer WD, Brown HA, Jiang X, Sternweis PC: Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. J Biol Chem. 1996, 271: 4504-4510. 10.1074/jbc.271.13.7412.View ArticlePubMedGoogle Scholar
- Bandhyopadhyay G, Sajan MP, Kanoh Y, Standaert ML, Quon MJ, Reed BC, Dikic I, Farese RV: Glucose activates protein kinase C-zeta/lambda through proline-rich tyrosine kinase-2, extracellular signal-regulated kinase, and phospholipase D: a novel mechanism for activating glucose transporter translocation. J Biol Chem. 2001, 276: 35537-35545. 10.1074/jbc.M106042200.View ArticleGoogle Scholar
- Melendez AJ, Harnett MM, Allen JM: Crosstalk between ARF6 and protein kinase C alpha in Fc(gamma)RI-mediated activation of phospholipase D1. Curr Biol. 2001, 11: 869-874. 10.1016/S0960-9822(01)00260-3.View ArticlePubMedGoogle Scholar
- Mwanjewe J, Spitaler M, Ebner M, Windegger M, Geiger M, Kampfer S, Hofmann J, Uberall F, Grunicke HH: Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-iota. Biochem J. 2001, 359: 211-217. 10.1042/0264-6021:3590211.PubMed CentralView ArticlePubMedGoogle Scholar
- Parmentier JH, Smelcer P, Pavicevic Z, Basic E, Idrizovic A, Estes A, Malik KU: PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. Hypertension. 2003, 41: 794-800. 10.1161/01.HYP.0000047873.76255.0B.View ArticlePubMedGoogle Scholar
- Pettitt TR, McDermott M, Saqib KM, Shimwell N, Wakelam MJO: Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells. Biochem J. 2001, 360: 707-715. 10.1042/0264-6021:3600707.PubMed CentralView ArticlePubMedGoogle Scholar
- Parmentier JH, Muthalif MM, Saeed AE, Malik KU: Phospholipase D activation by norepinephrine is mediated by 12(s)-, 15(s)-, and 20-hydroxyeicosatetraenoic acids generated by stimulation of cytosolic phospholipase A2. tyrosine phosphorylation of phospholipase D2 in response to norepinephrine. J Biol Chem. 2001, 276: 15704-15711. 10.1074/jbc.M011473200.View ArticlePubMedGoogle Scholar
- Standaert ML, Galloway L, Karnam P, Bandyopadhyay G, Moscat J, Farese RV: Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport. J Biol Chem. 1997, 272: 30075-30082. 10.1074/jbc.272.48.30075.View ArticlePubMedGoogle Scholar
- Ballou LM, Cross ME, Huang S, McReynolds EM, Zhang BX, Lin R: Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts. J Biol Chem. 2000, 275: 4803-4809. 10.1074/jbc.275.7.4803.View ArticlePubMedGoogle Scholar
- Paramio JM, Segrelles C, Ruiz S, Jorcano JL: Inhibition of protein kinase B (PKB) and PKC zeta mediates keratin K10-induced cell cycle arrest. Mol Cell Biol. 2001, 21: 7449-7459. 10.1128/MCB.21.21.7449-7459.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Chou MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, Newton AC, Schaffhausen BS, Toker A: Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol. 1998, 8: 1069-77.View ArticlePubMedGoogle Scholar
- Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ: Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science. 1998, 281: 2042-5. 10.1126/science.281.5385.2042.View ArticlePubMedGoogle Scholar
- Smith L, Smith JB: Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop. J Biol Chem. 2002, 277: 45866-45873. 10.1074/jbc.M206420200.View ArticlePubMedGoogle Scholar
- Le Good JA, Brindley DN: Molecular mechanisms regulating PKC-ζ turnover and cellular transformation. Biochem J. 2003,Google Scholar
- Diaz-Meco MT, Municio MM, Frutos S, Sanchez P, Lozano J, Sanz L, Moscat J: The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. Cell. 1996, 86: 777-786. 10.1016/S0092-8674(00)80152-X.View ArticlePubMedGoogle Scholar
- Diaz-Meco MT, Moscat J: MEK5: a new target of the atypical protein kinase C isoforms in mitogenic signaling. Mol Cell Biol. 2001, 21: 1218-1227. 10.1128/MCB.21.4.1218-1227.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Moscat J, Diaz-Meco MT: The atypical protein kinase Cs. Functional specificity mediated by specific protein adapters. EMBO Rep. 2000, 1: 399-403. 10.1093/embo-reports/kvd098.PubMed CentralView ArticlePubMedGoogle Scholar
- Susuki A, Akimoto K, Ohno S: Protein kinase C λ/ι (PKCλ/ι): A PKC isotype essential for the development of multicellular organisms. J Biochem. 2003, 133: 9-16. 10.1093/jb/mvg018.View ArticleGoogle Scholar
- Plant PJ, Fawcett JP, Lin DC, Holdorf AD, Binns K, Kulkarni S, Pawson T: A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl. Nat Cell Biol. 2003, 5: 301-308. 10.1038/ncb948.View ArticlePubMedGoogle Scholar
- Izumi Y, Hirose T, Tamai Y, Hirai S, Nagashima Y, Fujimoto T, Tabuse Y, Kemphues KJ, Ohno S: An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol. 1998, 143: 95-106. 10.1083/jcb.143.1.95.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.