title: (S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.
abstract: The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.
authors.forename: Aubin
authors.initials: A
authors.lastname: Moutal
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.
authors.forename: Liberty
authors.initials: L
authors.lastname: François-Moutal
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Perez-Miller
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.
authors.forename: Karissa
authors.initials: K
authors.lastname: Cottier
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.
authors.forename: Lindsey Anne
authors.initials: LA
authors.lastname: Chew
authors.affiliation: Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea.
authors.forename: Seul Ki
authors.initials: SK
authors.lastname: Yeon
authors.affiliation: Nuclear Magnetic Resonance Facility, Department of Chemistry and Biochemistry, College of Medicine, University of Arizona, Tucson, AZ, 85742, USA.
authors.forename: Jixun
authors.initials: J
authors.lastname: Dai
authors.affiliation: Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea.
authors.forename: Ki Duk
authors.initials: KD
authors.lastname: Park
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA. maykhanna@email.arizona.edu.
authors.forename: May
authors.initials: M
authors.lastname: Khanna
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA. rkhanna@email.arizona.edu.|Neuroscience Graduate Interdisciplinary Program, College of Medicine, University of Arizona, Tucson, AZ, 85742, USA. rkhanna@email.arizona.edu.
authors.forename: Rajesh
authors.identifier: http://orcid.org/0000-0002-9066-2969
authors.initials: R
authors.lastname: Khanna
mesh_terms: D000081:Acetamides / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D005260:Female; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism* / Q000494:pharmacology; D000078334:Lacosamide; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017207:Rats, Sprague-Dawley; D014404:Tubulin / Q000378:metabolism
chemical_list: D000081:Acetamides; C500963:Cacna1b protein, mouse; D020864:Calcium Channels, N-Type; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; C095114:collapsin response mediator protein-2; D000078334:Lacosamide; D051360:Cyclin-Dependent Kinase 5; D002118:Calcium
keywords: (S)-Lacosamide; CRMP2; CaV2.2; Cdk5; DSF; GSK-3β; STD NMR
references.citation: Biochemistry. 1992 Apr 28;31(16):3998-4003
references.pmid: 1567850
references.citation: Biochem Biophys Res Commun. 2003 May 30;305(2):392-9
references.pmid: 12745088
references.citation: Prog Brain Res. 1996;108:333-46
references.pmid: 8979812
references.citation: FEBS Lett. 2012 Nov 2;586(21):3813-8
references.pmid: 23022559
references.citation: J Neurochem. 2013 Jun;125(5):685-97
references.pmid: 23470087
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Aug;60(Pt 8):1355-63
references.pmid: 15272157
references.citation: Mol Neurobiol. 2015 Apr;51(2):599-609
references.pmid: 24944082
references.citation: Nat Neurosci. 2001 Aug;4(8):781-2
references.pmid: 11477421
references.citation: J Neurochem. 2007 May;101(4):906-17
references.pmid: 17250651
references.citation: Future Neurol. 2012 Nov 1;7(6):749-771
references.pmid: 23308041
references.citation: Cell Signal. 2009 Dec;21(12):1818-26
references.pmid: 19666111
references.citation: Nat Cell Biol. 2002 Aug;4(8):583-91
references.pmid: 12134159
references.citation: Trends Neurosci. 1988 Jul;11(7):315-21
references.pmid: 2465636
references.citation: J Neurochem. 2013 Mar;124(6):869-79
references.pmid: 23106100
references.citation: Neuron. 2006 Jun 15;50(6):897-909
references.pmid: 16772171
references.citation: J Neurochem. 2005 Sep;94(6):1535-45
references.pmid: 15992363
references.citation: Genes Cells. 2005 Feb;10 (2):165-79
references.pmid: 15676027
references.citation: Science. 1986 Jun 27;232(4758):1638-40
references.pmid: 3715470
references.citation: Anal Biochem. 2006 Oct 15;357(2):289-98
references.pmid: 16962548
references.citation: J Biol Chem. 2010 Aug 13;285(33):25296-307
references.pmid: 20538611
references.citation: Int J Cancer. 2013 May 1;132(9):1986-95
references.pmid: 23023514
references.citation: J Med Chem. 1996 Apr 26;39(9):1907-16
references.pmid: 8627614
references.citation: Front Cell Neurosci. 2014 May 28;8:135
references.pmid: 24904280
references.citation: Cell Calcium. 2010 Oct;48(4):183-94
references.pmid: 20888635
references.citation: J Biol Chem. 2008 Jun 27;283(26):18227-37
references.pmid: 18460467
references.citation: J Cell Sci. 2009 Dec 1;122(Pt 23):4351-62
references.pmid: 19903690
references.citation: Neuroscientist. 2014 Dec;20(6):589-98
references.pmid: 24402611
references.citation: J Neurosci. 2013 Nov 13;33(46):18298-310
references.pmid: 24227739
references.citation: J Neurosci. 2004 Oct 13;24(41):8994-9004
references.pmid: 15483118
references.citation: Mol Biol Cell. 2005 Jan;16(1):32-9
references.pmid: 15509652
references.citation: Mol Cell Biol. 2005 Nov;25(22):9920-35
references.pmid: 16260607
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Ernst Schering Res Found Workshop. 2004;(44):149-67
references.pmid: 14579779
references.citation: J Biol Chem. 2012 Oct 12;287(42):35065-77
references.pmid: 22891239
references.citation: J Mol Graph Model. 1999 Feb;17(1):57-61
references.pmid: 10660911
references.citation: Proc Natl Acad Sci U S A. 1986 Aug;83(16):6179-83
references.pmid: 3461482
references.citation: Exp Cell Res. 2009 Oct 1;315(16):2856-68
references.pmid: 19559021
references.citation: J Cell Sci. 2013 Sep 15;126(Pt 18):4262-73
references.pmid: 23868973
references.citation: J Neurochem. 1997 Dec;69(6):2261-9
references.pmid: 9375656
references.citation: J Neurosci. 2008 Mar 5;28(10):2366-74
references.pmid: 18322083
references.citation: Nat Cell Biol. 2011 Nov 06;13(12 ):1415-23
references.pmid: 22057101
references.citation: J Pharmacol Exp Ther. 2008 Jul;326(1):89-99
references.pmid: 18378801
references.citation: Epilepsia. 2013 Jul;54(7):1167-75
references.pmid: 23750855
references.citation: Hum Mol Genet. 2014 Aug 1;23 (15):4051-63
references.pmid: 24634145
references.citation: Arch Pharm Res. 2011 Dec;34(12):2059-64
references.pmid: 22210031
references.citation: J Alzheimers Dis. 2009;16(1):149-56
references.pmid: 19158430
references.citation: Nat Med. 2011 Jun 05;17(7):822-9
references.pmid: 21642979
references.citation: Nature. 1995 Dec 21-28;378(6559):785-9
references.pmid: 8524413
references.citation: Neuroscience. 2009 Jun 30;161(2):403-12
references.pmid: 19344753
references.citation: J Biol Chem. 2009 Nov 6;284(45):31375-90
references.pmid: 19755421
references.citation: J Comput Chem. 2010 Jan 30;31(2):455-61
references.pmid: 19499576
references.citation: Neurosci Res. 2013 Nov;77(3):180-5
references.pmid: 23994236
references.citation: Biophys J. 2000 Mar;78(3):1606-19
references.pmid: 10692345
references.citation: Dev Biol. 2000 Nov 15;227(2):581-94
references.pmid: 11071776
references.citation: Mol Neurobiol. 2003 Aug;28(1):51-64
references.pmid: 14514985
references.citation: Front Cell Neurosci. 2015 Jan 26;8:471
references.pmid: 25674050
references.citation: J Neurosci. 2010 Aug 11;30(32):10639-54
references.pmid: 20702696
references.citation: FEBS J. 2008 Sep;275(18):4583-96
references.pmid: 18699782
references.citation: J Neurosci. 1996 May 15;16(10):3236-46
references.pmid: 8627361
references.citation: Breast Cancer. 2014 Nov;21(6):715-23
references.pmid: 23381229
references.citation: Neuroscience. 2012 May 17;210:451-66
references.pmid: 22433297
references.citation: Ther Drug Monit. 2010 Aug;32(4):448-52
references.pmid: 20386357
references.citation: Cell. 2012 May 25;149(5):1112-24
references.pmid: 22632974
references.citation: J Neurochem. 2007 Nov;103(3):1132-44
references.pmid: 17683481
references.citation: Genes Dev. 1996 Apr 1;10 (7):816-25
references.pmid: 8846918
references.citation: Channels (Austin). 2013 May-Jun;7(3):153-9
references.pmid: 23510938
references.citation: J Comput Chem. 2005 Dec;26(16):1752-80
references.pmid: 16211539
references.citation: J Neurochem. 2005 Jun;93(6):1371-82
references.pmid: 15935053
references.citation: J Biol Chem. 2004 Nov 26;279(48):50176-80
references.pmid: 15466863
references.citation: J Biol Chem. 2006 Jun 16;281(24):16591-8
references.pmid: 16611631
references.citation: Nature. 1995 Aug 10;376(6540):509-14
references.pmid: 7637782
references.citation: PLoS One. 2012;7(2):e31242
references.pmid: 22363593
references.citation: Front Cell Neurosci. 2014 Jul 24;8:196
references.pmid: 25104922
references.citation: Cell. 2005 Jan 14;120(1):137-49
references.pmid: 15652488
references.citation: Angew Chem Int Ed Engl. 2003 Feb 24;42(8):864-90
references.pmid: 12596167