title: Cyclin-dependent kinase 5 promotes the stability of corneal epithelial cell junctions.
abstract: OBJECTIVE
  Although cyclin-dependent kinase 5 (Cdk5) inhibits the formation of junctions containing N-cadherin, the effect of Cdk5 on junctions containing E-cadherin is less clear. The present study investigates the functional significance of Cdk5 in forming and maintaining cell-cell stability in corneal epithelial cells.
  
  
  METHODS
  A Cdk5-deficient human corneal limbal epithelial cell line was generated by lentiviral transduction of small hairpin RNA specific for Cdk5 (shCdk5-HCLE cells). A blasticidin-inducible vector for expression of Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin-resistant cells were isolated for analysis. Cell aggregations were performed using HCLE, Cdk5 inhibitor olomoucine, ShCdk5, and MDA-MB 231 cells in the presence and absence of calcium, and particle size was measured using image analysis software. Relative protein concentrations were measured with immunoblotting and quantitative densitometry. Total internal reflection fluorescence (TIRF) microscopy was performed on cells transfected with green fluorescent protein (GFP)-E-cadherin or GFP-p120, and internalization of boundary-localized proteins was analyzed with particle tracking software. The stability of surface-exposed proteins was determined by measuring the recovery of biotin-labeled proteins with affinity chromatography. Rho and Rac activity was measured with affinity chromatography and immunoblotting.
  
  
  RESULTS
  Examining the effect of Cdk5 on E-cadherin containing epithelial cell-cell adhesions using a corneal epithelial cell line (HCLE), we found that Cdk5 and Cdk5 (pY15) coimmunoprecipitate with E-cadherin and Cdk5 (pY15) colocalizes with E-cadherin at cell-cell junctions. Inhibiting Cdk5 activity in HCLE or suppressing Cdk5 expression in a stable HCLE-derived cell line (ShHCLE) decreased calcium-dependent cell adhesion, promoted the cytoplasmic localization of E-cadherin, and accelerated the loss of surface-biotinylated E-cadherin. TIRF microscopy of GFP-E-cadherin in transfected HCLE cells showed an actively internalized sub-population of E-cadherin, which was not bound to p120 as it was trafficked away from the cell-cell boundary. This population increased in the absence of Cdk5 activity, suggesting that Cdk5 inhibition promotes dissociation of p120/E-cadherin junctional complexes. These effects of Cdk5 inhibition or suppression were accompanied by decreased Rac activity, increased Rho activity, and enhanced binding of E-cadherin to the Rac effector Ras GTPase-activating-like protein (IQGAP1). Cdk5 inhibition also reduced adhesion in a cadherin-deficient cell line (MDA-MB-231) expressing exogenous E-cadherin, although Cdk5 inhibition promoted adhesion when these cells were transfected with N-cadherin, as previous studies of Cdk5 and N-cadherin predicted. Moreover, Cdk5 inhibition induced N-cadherin expression and formation of N-cadherin/p120 complexes in HCLE cells.
  
  
  CONCLUSIONS
  These results indicate that loss of Cdk5 activity destabilizes junctional complexes containing E-cadherin, leading to internalization of E-cadherin and upregulation of N-cadherin. Thus, Cdk5 activity promotes stability of E-cadherin-based cell-cell junctions and inhibits the E-cadherin-to-N-cadherin switch typical of epithelial-mesenchymal transitions.
authors: National Eye Institute, NIH, Rockville, MD 20852, USA. arpithaparthasarathy@yahoo.com
authors: Parthasarathy
authors: P
authors: Arpitha
authors: Chun Y
authors: CY
authors: Gao
authors: Brajendra K
authors: BK
authors: Tripathi
authors: Senthil
authors: S
authors: Saravanamuthu
authors: Peggy
authors: P
authors: Zelenka
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D058750:Epithelial-Mesenchymal Transition; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins; D034741:RNA, Small Interfering; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human
references: Cancer Biol Ther. 2005 Jan;4(1):28-31
references: 15662113
references: J Cell Biol. 2004 Aug 2;166(3):393-405
references: 15277544
references: EMBO Rep. 2003 Jun;4(6):571-4
references: 12776176
references: Neuron. 2000 Jun;26(3):633-46
references: 10896159
references: Trends Cell Biol. 2006 May;16(5):242-9
references: 16595175
references: Mol Biol Cell. 2001 Sep;12(9):2711-20
references: 11553710
references: Neuron. 2000 Dec;28(3):681-96
references: 11163259
references: Oncogene. 2006 Jan 5;25(1):8-19
references: 16170364
references: J Biol Chem. 2006 Feb 24;281(8):5288-99
references: 16361708
references: J Cell Sci. 2004 Aug 15;117(Pt 18):4089-98
references: 15280426
references: Nat Cell Biol. 2002 Mar;4(3):222-31
references: 11836526
references: J Biol Chem. 2008 Jun 27;283(26):18344-54
references: 18407999
references: J Cell Biol. 2002 Nov 11;159(3):465-76
references: 12427869
references: Exp Cell Res. 2008 Oct 15;314(17):3187-97
references: 18773890
references: Cell Mol Life Sci. 2011 Oct;68(20):3425-36
references: 21442427
references: J Cell Sci. 1992 Sep;103 ( Pt 1):233-43
references: 1385459
references: Biochim Biophys Acta. 2007 Jan;1773(1):2-7
references: 17175391
references: J Cell Biol. 2006 Oct 9;175(1):135-46
references: 17015620
references: J Cell Biol. 2003 Nov 10;163(3):547-57
references: 14610057
references: J Cell Sci. 2002 May 15;115(Pt 10):2109-17
references: 11973352
references: Oncogene. 2012 Jan 19;31(3):376-89
references: 21685945
references: Curr Opin Cell Biol. 2001 Oct;13(5):604-10
references: 11544030
references: Mol Cell Biol. 2009 Dec;29(24):6488-99
references: 19822667
references: J Cell Sci. 2005 May 15;118(Pt 10):2085-92
references: 15890984
references: Invest Ophthalmol Vis Sci. 2003 Jun;44(6):2496-506
references: 12766048
references: J Cell Biol. 2000 May 15;149(4):775-82
references: 10811819
references: Biochim Biophys Acta. 2007 Jan;1773(1):34-46
references: 17028013
references: Mol Cell Biol. 2005 Jan;25(1):389-402
references: 15601859
references: J Biol Chem. 2008 Feb 22;283(8):5014-22
references: 18089563
references: J Natl Cancer Inst. 1974 Sep;53(3):661-74
references: 4412247
references: J Cell Biol. 1989 Dec;109(6 Pt 1):2809-16
references: 2687287
references: J Cell Biol. 2000 Aug 7;150(3):567-80
references: 10931868
references: Curr Biol. 2000 Apr 6;10(7):363-72
references: 10753743
references: EMBO J. 2002 Apr 15;21(8):1948-56
references: 11953314
references: Semin Cell Dev Biol. 2008 Apr;19(2):113-24
references: 18035561
references: Bioessays. 2006 Oct;28(10):1023-34
references: 16998837
references: Nature. 1994 Sep 29;371(6496):419-23
references: 8090221
references: J Biol Chem. 2008 Jan 18;283(3):1692-1704
references: 17984089
references: Nat Cell Biol. 2012 Aug;14(8):818-828
references: 22750944
references: Invest Ophthalmol Vis Sci. 2012 Aug 07;53(9):5139-53
references: 22736606
references: Curr Opin Cell Biol. 2004 Oct;16(5):522-7
references: 15363802
references: J Cell Biol. 2008 Nov 17;183(4):737-49
references: 19015320
references: J Cell Sci. 2000 Mar;113 ( Pt 6):975-83
references: 10683146
references: J Biol Chem. 2008 Feb 22;283(8):5127-37
references: 18057010
references: Biochim Biophys Acta. 2007 Jan;1773(1):8-16
references: 16949165
references: J Biol Chem. 2010 Sep 17;285(38):29491-501
references: 20595387
references: J Cell Biol. 1998 May 4;141(3):779-89
references: 9566976
references: Hum Mol Genet. 2009 Oct 1;18(19):3632-44
references: 19584084
references: Dev Genet. 1997;20(3):267-75
references: 9216066
references: Mol Cell Biol. 2004 Apr;24(7):2808-19
references: 15024070