title: Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14.
abstract: Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca(2+) and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis of SCA14.
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan ; Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University Kumamoto, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Seki
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan ; Biosignal Research Center, Kobe University Kobe, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Biosignal Research Center, Kobe University Kobe, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Hide
authors.affiliation: Biosignal Research Center, Kobe University Kobe, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Sakai
keywords: MARCKS; actin cytoskeleton; macropinocytosis; spinocerebellar ataxia type 14; translocation; γPKC
references.citation: Am J Hum Genet. 2003 Apr;72(4):839-49
references.pmid: 12644968
references.citation: J Biol Chem. 2013 Jun 14;288(24):17145-55
references.pmid: 23625928
references.citation: J Cell Biol. 1997 Dec 15;139(6):1465-76
references.pmid: 9396752
references.citation: J Biol Chem. 2006 Aug 4;281(31):22321-22331
references.pmid: 16751194
references.citation: J Biochem. 2002 Nov;132(5):663-8
references.pmid: 12417013
references.citation: PLoS One. 2012;7(2):e31232
references.pmid: 22363588
references.citation: Trends Cell Biol. 2009 Jul;19(7):317-24
references.pmid: 19540123
references.citation: Arch Neurol. 2002 Aug;59(8):1291-5
references.pmid: 12164726
references.citation: Neurology. 2005 Apr 12;64(7):1258-60
references.pmid: 15824357
references.citation: J Cell Sci. 2008 Jul 15;121(Pt 14):2339-49
references.pmid: 18577575
references.citation: FASEB J. 2001 Jul;15(9):1634-6
references.pmid: 11427510
references.citation: Jpn J Pharmacol. 1998 Dec;78(4):411-7
references.pmid: 9920197
references.citation: Traffic. 2002 Mar;3(3):186-92
references.pmid: 11886589
references.citation: J Cell Sci. 2000 Jul;113 ( Pt 14):2575-84
references.pmid: 10862715
references.citation: J Neurosci. 1988 Feb;8(2):369-82
references.pmid: 3276831
references.citation: J Biol Chem. 2003 Dec 26;278(52):52747-54
references.pmid: 14527960
references.citation: J Neurosci. 2009 Aug 26;29(34):10488-98
references.pmid: 19710302
references.citation: Mov Disord. 2006 Sep;21(9):1355-60
references.pmid: 16763984
references.citation: Annu Rev Biochem. 2009;78:857-902
references.pmid: 19317650
references.citation: Genes Cells. 2010 May;15(5):425-38
references.pmid: 20398063
references.citation: J Cell Biol. 2000 Jun 26;149(7):1455-72
references.pmid: 10871285
references.citation: Am J Physiol. 1999 Dec;277(6):C1239-49
references.pmid: 10600776
references.citation: J Neurochem. 2002 Nov;83(3):490-503
references.pmid: 12390511
references.citation: Am J Physiol. 1999 Jul;277(1):C163-73
references.pmid: 10409119
references.citation: Cell Mol Life Sci. 2007 Feb;64(3):263-70
references.pmid: 17180302
references.citation: Nature. 1992 Apr 16;356(6370):618-22
references.pmid: 1560845
references.citation: Traffic. 2008 Sep;9(9):1437-45
references.pmid: 18532987
references.citation: Arch Biochem Biophys. 1998 Nov 15;359(2):151-9
references.pmid: 9808756
references.citation: Nat Rev Mol Cell Biol. 2010 Feb;11(2):103-12
references.pmid: 20094051
references.citation: Brain. 2005 Feb;128(Pt 2):436-42
references.pmid: 15618281
references.citation: J Pharmacol Sci. 2011;116(3):239-47
references.pmid: 21666345
references.citation: Genes Cells. 2005 Mar;10(3):225-39
references.pmid: 15743412
references.citation: Neurochem Int. 2008 Nov;53(5):155-64
references.pmid: 18691623
references.citation: J Biol Chem. 2008 Jul 11;283(28):19854-63
references.pmid: 18499672
references.citation: Neurobiol Dis. 2009 Feb;33(2):260-73
references.pmid: 19041943
references.citation: Mol Cell Neurosci. 2009 Jan;40(1):27-38
references.pmid: 18848894
references.citation: Handb Clin Neurol. 2012;103:555-9
references.pmid: 21827914
references.citation: Int J Med Microbiol. 2002 Feb;291(6-7):487-94
references.pmid: 11890548
references.citation: Mol Biol Cell. 2008 Nov;19(11):4804-13
references.pmid: 18799624
references.citation: Immunol Cell Biol. 2011 Nov;89(8):836-43
references.pmid: 21423264
references.citation: Trends Cell Biol. 1995 Nov;5(11):424-8
references.pmid: 14732047
references.citation: J Biol Chem. 2005 Aug 12;280(32):29096-106
references.pmid: 15964845
references.citation: Nat Cell Biol. 2004 Jul;6(7):585-91
references.pmid: 15232591
references.citation: J Cell Sci. 1999 Aug;112 ( Pt 15):2549-57
references.pmid: 10393811
references.citation: Eur J Neurosci. 2007 Dec;26(11):3126-40
references.pmid: 18005063