This article has Open Peer Review reports available.
Protein kinase Cepsilon is important for migration of neuroblastoma cells
© Stensman and Larsson; licensee BioMed Central Ltd. 2008
Received: 20 May 2008
Accepted: 11 December 2008
Published: 11 December 2008
Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.
PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.
Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.
PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration.
Cell migration plays a central role in a wide range of different biological processes, both normal and pathological, including wound healing, inflammatory response and tumour metastasation . The capacity of cells to migrate is dependent on signals from the extracellular environment which are transduced via networks of intracellular signal transduction proteins. A variety of intracellular signalling molecules including members of the protein kinase C (PKC) family of isoforms participate in the regulation of cellular migration [2–5].
PKC comprises a family of related serine/threonine kinases that are involved in a number of cellular processes such as proliferation and apoptosis in addition to their roles in regulating cellular morphology, adhesion and migration. Based on regulatory and structural properties, the PKC isoforms can be grouped in three different subfamilies; the classical PKCs (α, βI, βII and γ) are activated by Ca2+, phospholipids and diacylglycerol (DAG), the novel PKCs (δ, ε, η and θ) are activated by phospholipids and DAG but are insensitive to Ca2+ while the atypical PKCs (ζ and ι/λ) require neither DAG nor Ca2+ for activation .
An important role for PKC in cell migration has long been suggested for a wide range of cell types by the fact that phorbol esters, which are general PKC activators, enhance the motility of these cells [7–9]. Further studies have failed to pinpoint one or a few particular isoforms as being general regulators of migration . It rather seems as if many isoforms have the capacity to influence the migratory behaviour and which isoform that is involved depends on the cell type. Overexpression of PKCα has been shown to increase motility in MCF-10 cells , 2C4 fibrosarcoma cells  and the breast cancer cell lines MCF-7  and MDA-MB-435  and PKCβI can mediate cytoskeletal rearrangements and platelet spreading on fibrinogen . Activation of PKCδ with epidermal growth factor is important for migration of fibroblasts  and elevated levels of PKCδ contribute to a more metastatic phenotype of mammary tumour cells . Finally, PKCε has been suggested to be important for glioma cell migration  and inhibition of PKCε leads to decreased motility of fibroblasts  and head and neck squamous cell carcinoma .
Neuroblastoma is the most common extracranial solid tumour among pediatric cancers affecting approximately 1 in 7000 live births . The cancer is frequently lethal and this is coupled to widespread metastasation. It would therefore be beneficial to understand what regulates the migratory behaviour, which is one precondition for infiltration and spread, of neuroblastoma cells. This study was designed to investigate whether PKC isoforms can influence the migratory capacity of neuroblastoma cells and to elucidate putative pathways mediating the PKC effect.
Human SK-N-BE(2)C, KCN-69c and SH-SY5Y neuroblastoma cells were maintained in Minimal Essential Medium (Gibco) supplemented with 10% foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco).
Transfections with siRNA
Cell migration was assayed in triplicates using a 48-well transwell setup (Neuroprobe) using polycarbonate Nucleopore filters with 8 μm pore size. The underside of the membrane was precoated with 20 μg/ml fibronectin (Sigma) in PBS for 16 h at 4°C. Cells were dissociated with trypsin (Gibco) for 5 min followed by addition of 0.1% soy bean trypsin inhibitor (Invitrogen). Cells were centrifuged, resuspended in serum-free medium and 15,000 cells were seeded in the upper chamber of each well. The lower chambers contained serum-free medium supplemented with activators or inhibitors at the following concentrations: 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma), 16 nM; GF109203X and Gö6976, 2 μM (both Calbiochem); LY333531, 200 nM (Alexis); PD98059, 50 μM and LY294002, 20 μM (both Sigma). Cells were incubated for 6 h in 37°C. Non-migrated cells on the upper side of the membrane were removed by scraping, while migrated cells attached to the underside of the membrane were fixed for 10 min in methanol and stained with Vectashield with DAPI (Vector laboratories). Cells were examined using a fluorescence microscope and all cells in a specified area in the middle of the membrane were counted.
Cells were seeded at a density of 450,000 cells per well in 12-well cell culture plates. After incubation for 24 hours, the confluent cell monolayer was scraped with a pipette tip creating a scratch in each well. Medium containing serum supplemented with TPA or inhibitors was added and cells were incubated at 37°C. For experiments with siRNA, 70,000 cells were seeded in 12-well cell culture plates and treated with siRNA as described and 18 hours after the last transfection, cell monolayers were scratched. Cells were photographed at different time points and the scratch area was measured using ImageJ.
1.0 × 106 cells were seeded in 60-mm cell culture dishes and incubated for 24 hours. Cells were pre-incubated for 1 h in serum-free medium prior to stimulation. Cells were washed twice in PBS and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.2, 160 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA) containing 40 μl/ml protease inhibitors (Roche Applied Science). Cells transfected with siRNA were lysed in the same way 18 h after the last transfection. Lysates were centrifuged for 10 min at 14,000 × g at 4°C. Proteins were electrophoretically separated on a 10% NuPAGE Novex Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene diflouride membrane (Millipore). For detection, membranes were incubated with primary antibodies against phospho-MARCKS (1:500), phospho-Erk (1:500), Erk (1:500) (all Cell Signaling), MARCKS (1:1000) (Upstate), PKCα (1:3000), PKCβII (1:500), PKCδ (1:500) or PKCε (1:500) (all Santa Cruz Biotechnology) followed by incubation with a horseradish peroxidase-labelled secondary antibody (1:5000) (Amersham Biosciences). Horseradish peroxidase was thereafter visualised using the SuperSignal system (Pierce) as substrate. The chemoluminescence was detected with a CCD camera (Fujifilm).
Calculations and statistics
IC50 values were calculated by doing a curve fit analysis to the equation y = A/(1+x/B) where A is the maximal effect and B is the IC50 value. Statistical analyses were done by doing ANOVA followed by Duncan's multiple range test using p < 0.05 as level of for significance.
Activation of PKC stimulates migration of neuroblastoma cells
To investigate a putative role of PKC in neuroblastoma cell motility, the migration of SK-N-BE(2)C neuroblastoma cells was studied using transwell and scratch assays.
PKCε is necessary for SK-N-BE(2)C cell migration
SK-N-BE(2)C cells transfected with siRNAs were seeded in the upper wells of the transwell migration chambers and were allowed to migrate towards serum-free medium (Fig 4B) or medium supplemented with 16 nM TPA (Fig 4C). In both cases, treatment with the PKCε siRNA resulted in suppressed migration. Reduction of PKCα or PKCδ levels did not significantly influence migration.
Neither the PI3K pathway nor the Erk pathway is involved in PKCε-induced migration
The fact that the PD98059 caused a tendency to reduced migration in the scratch assay led us to investigate whether Erk is a mediator of the pro-migratory effect of PKCε. However, TPA induced Erk phosphorylation to the same extent in control cells as in cells with downregulated PKCε (Fig 6C), indicating that Erk is not a crucial mediator of the PKCε effect.
PKC-mediated phosphorylation of MARCKS
Cells were also transfected with siRNA oligos against PKCα, PKCδ and PKCε and stimulated with TPA for 1 h followed by analysis of MARCKS phosphorylation (Fig 7B). TPA treatment led to increased phosphorylation of MARCKS under all conditions indicating that several isoforms phosphorylate MARCKS in SK-N-BE(2)C cells.
A major problem in curing cancer is the capacity of cancer cells to migrate, invade tissues and subsequently seed metastases in other organs. This is also the case for neuroblastoma, a pediatric cancer derived from the peripheral sympathetic nervous system. The mechanisms determining the migratory capacity of neuroblastoma cells are not fully understood. Several reports indicate that growth factors, such as IGF-1  and PDGF , and integrins  can stimulate neuroblastoma cell motility. In this study we demonstrate that a direct activation of PKC is sufficient to induce migration of neuroblastoma cells and PKC thus arises as an interesting target to suppress the motility of these cells.
Activation of PKC stimulated migration of two different neuroblastoma cell lines, SK-N-BE(2)C and KCN-69c, whereas the SH-SY5Y cell line did not increase its motility in response to PKC activators. This is not due to a poor migratory capacity of these cells since they migrate in response to other stimuli [25, 27, 28]. However, in terms of PKC effects SH-SY5Y cells are unique in that they differentiate upon treatment with TPA  which may explain why they do not migrate upon PKC activation. Another possible explanation is the fact that SK-N-BE(2)C and KCN-69c, but not SH-SY5Y cells, carry an NMYC amplification which results in more aggressive tumours . The amplification may be associated with the presence of a pathway that transduces a PKC signal to increased motility. However, a larger panel of neuroblastoma cells is necessary to corroborate such a hypothesis.
PKC comprises a family of ten related isoforms, eight of which are TPA-sensitive, and of these, neuroblastoma cells generally express PKCα, PKCβII, PKCδ and PKCε . Reducing the levels of PKCε, but not of PKCα or PKCδ, with siRNA inhibited migration both under basal conditions and when cells were stimulated with TPA. This is not due to off-target effects since three different siRNA oligonucleotides against PKCε all led to a reduced migration. Despite transfecting the cells with siRNA for three consecutive days we were not able to reduce the levels of PKCε completely which raises the possibility that even more suppressive effects could be obtained if PKCε could be depleted from the cells. A role of PKCε is in line with the suppression of the TPA effect obtained by the general PKC inhibitor GF109203X. However, in contrast to PKCε siRNA treatment, the kinase inhibitor did not affect migration under basal conditions. PKCε has been shown to induce morphological effects, induction of neurites  and dismantling of stress fibres , independently of its kinase activity. Our results indicate that also some of the promigratory effects of PKCε may be exerted independently of its catalytic activity.
The inhibitor of classical PKCs, Gö6976, also suppressed migration, indicating a potential role for these isoforms in migration. However, Gö6976 influenced migration both in the absence and presence of TPA contrasting the effect of GF109203X, which did not have an effect under basal conditions. Gö6976 has been shown to exert effects that are unrelated to and independent of PKC inhibition [34–36]. Furthermore, neither inhibition of PKCα with siRNA nor of PKCβ with LY333531 suppressed migration. This makes it more conceivable that PKCε is the primary promigratory PKC isoform in neuroblastoma cells and that Gö6976 inhibits motility by some other actions.
There are several different mechanisms through which PKCε may mediate its effects on cellular motility. Integrins are receptors for extracellular matrix components and are critically involved in the regulation of cell motility. PKCε has been shown to both regulate the recycling of integrins [18, 37] and participate in down stream signalling following integrin clustering . One of the putative PKCε targets is Erk which is targeted to focal adhesions following direct activation of PKC  or to focal complexes during HGF-mediated cell movement . Both of these events are mediated via PKCε but our data do not support a critical role of Erk in PKCε-mediated migration of neuroblastoma cells. Although there was a tendency towards suppression of the wound healing by PD98059, it had no effect in the transwell assay and downregulation of PKCε to levels that cause a reduced migration did not influence TPA-stimulated Erk phosphorylation.
In addition to regulating other signalling proteins, PKC can also phosphorylate several proteins, such as MARCKS and ERM proteins [11, 40], that more directly regulate the structure of the cytoskeleton. There was indeed a substantial PKC-mediated increase in MARCKS phosphorylation concomitant with TPA-stimulated migration indicating a role for MARCKS in the PKC-mediated motility of neuroblastoma cells. An involvement of MARCKS in PKC-regulated migration has been suggested in many other cell types [15, 41, 42] and our data would further support the general importance of this pathway.
However, experiments with siRNA showed that the phosphorylation of MARCKS was not altered when any of the isoforms PKCα, PKCδ or PKCε was downregulated. Since downregulation of PKCε leads to suppressed migration it does not seem as if MARCKS is specific and critical in the PKCε pathway. Instead it is conceivable that several isoforms phosphorylate MARCKS upon addition of TPA. This is further supported by the finding that the inhibitor of classical isoforms, Gö6976, partially reduces the phosphorylation whereas the general PKC inhibitor GF109203X has an even larger effect. MARCKS has been shown to be a high affinity substrate for both novel and classical PKC isoforms in vitro and in intact cells [43, 44] supporting our finding that several PKC isoforms can phosphorylate MARCKS in SK-N-BE(2)C cells.
In conclusion, we show for the first time that PKCε is necessary to promote migration of SK-N-BE(2)C neuroblastoma cells making it a possible target for blocking the motility of these cells.
This work was supported by grants from The Swedish Cancer Society, The Swedish Research Council, The Children's Cancer Foundation of Sweden, Malmö University Hospital Research Funds, and the Kock, Crafoord, Ollie and Elof Ericsson and Gunnar Nilsson Foundations.
- Lauffenburger DA, Horwitz AF: Cell migration: a physically integrated molecular process. Cell. 1996, 84: 359-369. 10.1016/S0092-8674(00)81280-5.View ArticlePubMedGoogle Scholar
- Friedl P, Wolf K: Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer. 2003, 3: 362-374. 10.1038/nrc1075.View ArticlePubMedGoogle Scholar
- Gopalakrishna R, Barsky SH: Tumor promoter-induced membrane-bound protein kinase C regulates hematogenous metastasis. Proc Natl Acad Sci USA. 1988, 85: 612-616. 10.1073/pnas.85.2.612.View ArticlePubMedPubMed CentralGoogle Scholar
- Hall A: Rho GTPases and the actin cytoskeleton. Science. 1998, 279: 509-514. 10.1126/science.279.5350.509.View ArticlePubMedGoogle Scholar
- Larsson C: Protein kinase C and the regulation of the actin cytoskeleton. Cell Signal. 2006, 18: 276-284. 10.1016/j.cellsig.2005.07.010.View ArticlePubMedGoogle Scholar
- Newton AC: Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev. 2001, 101: 2353-2364. 10.1021/cr0002801.View ArticlePubMedGoogle Scholar
- Defilippi P, Venturino M, Gulino D, Duperray A, Boquet P, Fiorentini C, Volpe G, Palmieri M, Silengo L, Tarone G: Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation. J Biol Chem. 1997, 272: 21726-21734. 10.1074/jbc.272.35.21726.View ArticlePubMedGoogle Scholar
- Rigot V, Lehmann M, Andre F, Daemi N, Marvaldi J, Luis J: Integrin ligation and PKC activation are required for migration of colon carcinoma cells. J Cell Sci. 1998, 111: 3119-3127.PubMedGoogle Scholar
- Varani J, Fligiel SE, Perone P: Directional motility in strongly malignant murine tumor cells. Int J Cancer. 1985, 35: 559-564. 10.1002/ijc.2910350422.View ArticlePubMedGoogle Scholar
- Sun XG, Rotenberg SA: Overexpression of protein kinase Cα in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation, and motility. Cell Growth Differ. 1999, 10: 343-352.PubMedGoogle Scholar
- Ng T, Parsons M, Hughes WE, Monypenny J, Zicha D, Gautreau A, Arpin M, Gschmeissner S, Verveer PJ, Bastiaens PIH, Parker PJ: Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J. 2001, 20: 2723-2741. 10.1093/emboj/20.11.2723.View ArticlePubMedPubMed CentralGoogle Scholar
- Ng T, Shima D, Squire A, Bastiaens PIH, Gschmeissner S, Humphries MJ, Parker PJ: PKCα regulates β1 integrin-dependent cell motility through association and control of integrin traffic. EMBO J. 1999, 18: 3909-3923. 10.1093/emboj/18.14.3909.View ArticlePubMedPubMed CentralGoogle Scholar
- Tan M, Li P, Sun M, Yin G, Yu D: Upregulation and activation of PKCα by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKCα and Src inhibitors. 2006, 25: 3286-3295.Google Scholar
- Buensuceso CS, Obergfell A, Soriani A, Eto K, Kiosses WB, Arias-Salgado EG, Kawakami T, Shattil SJ: Regulation of outside-in signaling in platelets by integrin-associated protein kinase Cβ. J Biol Chem. 2005, 280: 644-653.View ArticlePubMedGoogle Scholar
- Iwabu A, Smith K, Allen FD, Lauffenburger DA, Wells A: Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C δ-dependent pathway. J Biol Chem. 2004, 279: 14551-14560. 10.1074/jbc.M311981200.View ArticlePubMedGoogle Scholar
- Kiley SC, Clark KJ, Goodnough M, Welch DR, Jaken S: Protein kinase C delta involvement in mammary tumor cell metastasis. Cancer Res. 1999, 59: 3230-3238.PubMedGoogle Scholar
- Besson A, Wilson TL, Yong VW: The anchoring protein RACK1 links protein kinase Cε to integrin βchains. Requirment for adhesion and motility. J Biol Chem. 2002, 277: 22073-22084. 10.1074/jbc.M111644200.View ArticlePubMedGoogle Scholar
- Ivaska J, Whelan RDH, Watson R, Parker PJ: PKCε controls the traffic of β1 integrins in motile cells. EMBO J. 2002, 21: 3608-3619. 10.1093/emboj/cdf371.View ArticlePubMedPubMed CentralGoogle Scholar
- Pan Q, Bao LW, Teknos TN, Merajver SD: Targeted disruption of protein kinase Cε reduces cell invasion and motility through inactivation of RhoA and RhoC GTPases in head and neck squamous cell carcinoma. Cancer Res. 2006, 66: 9379-9384. 10.1158/0008-5472.CAN-06-2646.View ArticlePubMedPubMed CentralGoogle Scholar
- Brodeur GM: Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003, 3: 203-216. 10.1038/nrc1014.View ArticlePubMedGoogle Scholar
- Zeidman R, Trollér U, Raghunath A, Påhlman S, Larsson C: Protein kinase Cε actin-binding site is important for neurite outgrowth during neuronal differentiation. Mol Biol Cell. 2002, 13: 12-24. 10.1091/mbc.01-04-0210.View ArticlePubMedPubMed CentralGoogle Scholar
- Ling M, Troller U, Zeidman R, Stensman H, Schultz A, Larsson C: Identification of conserved amino acids N-terminal of the PKCεC1b domain crucial for protein kinase Cε-mediated induction of neurite outgrowth. J Biol Chem. 2005, 280: 17910-17919. 10.1074/jbc.M412036200.View ArticlePubMedGoogle Scholar
- Colon-Gonzalez F, Kazanietz MG: C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim Biophys Acta. 2006, 1761: 827-837.View ArticlePubMedGoogle Scholar
- Kim B, Feldman EL: Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization. J Neurochem. 1998, 71: 1333-1336.View ArticlePubMedGoogle Scholar
- Pola S, Cattaneo MG, Vicentini LM: Anti-migratory and anti-invasive effect of somatostatin in human neuroblastoma cells: Involvement of Rac and MAP kinase activity. J Biol Chem. 2003, 278: 40601-40606. 10.1074/jbc.M306510200.View ArticlePubMedGoogle Scholar
- Arbuzova A, Schmitz AA, Vergeres G: Cross-talk unfolded: MARCKS proteins. Biochem J. 2002, 362: 1-12. 10.1042/0264-6021:3620001.View ArticlePubMedPubMed CentralGoogle Scholar
- Meyer GE, Shelden E, Kim B, Feldman EL: Insulin-like growth factor I stimulates motility in human neuroblastoma cells. Oncogene. 2001, 20: 7542-7550. 10.1038/sj.onc.1204927.View ArticlePubMedGoogle Scholar
- Meyer A, van Golen CM, Kim B, van Golen KL, Feldman EL: Integrin expression regulates neuroblastoma attachment and migration. Neoplasia. 2004, 6: 332-342. 10.1593/neo.03445.View ArticlePubMedPubMed CentralGoogle Scholar
- Påhlman S, Odelstad L, Larsson E, Grotte G, Nilsson K: Phenotypic changes of human neuroblastoma cells in culture induced by 12-O-tetradecanoyl-phorbol-13-acetate. Int J Cancer. 1981, 28: 583-589. 10.1002/ijc.2910280509.View ArticlePubMedGoogle Scholar
- Corvi R, Savelyeva L, Schwab M: Patterns of oncogene activation in human neuroblastoma cells. J Neurooncol. 1997, 31: 25-31. 10.1023/A:1005721027709.View ArticlePubMedGoogle Scholar
- Zeidman R, Pettersson L, Ranga PS, Truedsson E, Fagerström S, Påhlman S, Larsson C: Novel and classical isoforms have different functions in proliferation, survival and differentiation of neuroblastoma cells. Int J Cancer. 1999, 81: 494-501. 10.1002/(SICI)1097-0215(19990505)81:3<494::AID-IJC26>3.0.CO;2-L.View ArticlePubMedGoogle Scholar
- Zeidman R, Löfgren B, Påhlman S, Larsson C: PKCε, via its regulatory domain and independently of its catalytic domain, induces neurite-like processes in neuroblastoma cells. J Cell Biol. 1999, 145: 713-726. 10.1083/jcb.145.4.713.View ArticlePubMedPubMed CentralGoogle Scholar
- Ling M, Trollér U, Zeidman R, Lundberg C, Larsson C: Induction of neurites by the regulatory domain of PKCδ and ε in neural cells is counteracted by PKC catalytic activity and the RhoA pathway. Exp Cell Res. 2004, 292: 135-150. 10.1016/j.yexcr.2003.08.013.View ArticlePubMedGoogle Scholar
- Beltman J, McCormick F, Cook SJ: The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem. 1996, 271: 27018-27024. 10.1074/jbc.271.43.27018.View ArticlePubMedGoogle Scholar
- Chen J, Lu G, Wang QJ: Protein kinase C-independent effects of protein kinase D3 in glucose transport in L6 myotubes. Mol Pharmacol. 2005, 67: 152-162. 10.1124/mol.104.004200.View ArticlePubMedGoogle Scholar
- Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000, 351: 95-105. 10.1042/0264-6021:3510095.View ArticlePubMedPubMed CentralGoogle Scholar
- Ivaska J, Vuoriluoto K, Huovinen T, Izawa I, Inagaki M, Parker PJ: PKCε-mediated phosphorylation of vimentin controls integrin recycling and motility. EMBO J. 2005, 24: 3834-3845. 10.1038/sj.emboj.7600847.View ArticlePubMedPubMed CentralGoogle Scholar
- Besson A, Davy A, Robbins SM, Yong VW: Differential activation of ERKs to focal adhesions by PKC ε is required for PMA-induced adhesion and migration of human glioma cells. Oncogene. 2001, 20: 7398-7407. 10.1038/sj.onc.1204899.View ArticlePubMedGoogle Scholar
- Kermorgant S, Zicha D, Parker PJ: PKC controls HGF-dependent c-Met traffic, signalling and cell migration. EMBO J. 2004, 23: 3721-3734. 10.1038/sj.emboj.7600396.View ArticlePubMedPubMed CentralGoogle Scholar
- Wu WC, Walaas SI, Nairn AC, Greengard P: Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes. Proc Natl Acad Sci USA. 1982, 79: 5249-5253. 10.1073/pnas.79.17.5249.View ArticlePubMedPubMed CentralGoogle Scholar
- Disatnik M-H, Boutet SC, Pacio W, Chan AY, Ross LB, Lee CH, Rando TA: The bi-directional translocation of MARCKS between membrane and cytosol regulates integrin-mediated muscle cell spreading. J Cell Sci. 2004, 117: 4469-4479. 10.1242/jcs.01309.View ArticlePubMedGoogle Scholar
- Myat MM, Anderson S, Allen LA, Aderem A: MARCKS regulates membrane ruffling and cell spreading. Curr Biol. 1997, 7: 611-614. 10.1016/S0960-9822(06)00262-4.View ArticlePubMedGoogle Scholar
- Fujise A, Mizuno K, Ueda Y, Osada S, Hirai S, Takayanagi A, Shimizu N, Owada MK, Nakajima H, Ohno S: Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate. J Biol Chem. 1994, 269: 31642-31648.PubMedGoogle Scholar
- Überall F, Giselbrecht S, Hellbert K, Fresser F, Bauer B, Gschwendt M, Grunicke HH, Baier G: Conventional PKC-α, novel PKC-ε and PKC-θ, but not atypical PKC-λ are MARCKS kinases in intact NIH 3T3 fibroblasts. J Biol Chem. 1997, 272: 4072-4078. 10.1074/jbc.272.7.4072.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/8/365/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.