title: Intermolecular autophosphorylation regulates myosin IIIa activity and localization in parallel actin bundles.
abstract: Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
authors.forename: Omar A
authors.initials: OA
authors.lastname: Quintero
authors.forename: Judy E
authors.initials: JE
authors.lastname: Moore
authors.forename: William C
authors.initials: WC
authors.lastname: Unrath
authors.forename: Uri
authors.initials: U
authors.lastname: Manor
authors.forename: Felipe T
authors.initials: FT
authors.lastname: Salles
authors.forename: M'hamed
authors.initials: M
authors.lastname: Grati
authors.forename: Bechara
authors.initials: B
authors.lastname: Kachar
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yengo
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018069:Hair Cells, Vestibular / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D025102:Myosin Type III / Q000235:genetics / Q000378:metabolism*; D009925:Organ of Corti / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000199:Actins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C491724:MYO3A protein, human; D025102:Myosin Type III; D018995:Myosin Heavy Chains
references.citation: Curr Opin Cell Biol. 2005 Apr;17(2):197-202
references.pmid: 15780597
references.citation: Methods Enzymol. 1982;85 Pt B:164-81
references.pmid: 7121269
references.citation: Nucleic Acids Res. 2002 Jan 15;30(2):E9
references.pmid: 11788735
references.citation: Science. 1993 Nov 12;262(5136):1038-42
references.pmid: 8235618
references.citation: Mol Biol Cell. 2003 Mar;14(3):1058-73
references.pmid: 12631723
references.citation: Nat Cell Biol. 2002 Mar;4(3):246-50
references.pmid: 11854753
references.citation: J Cell Biol. 1999 Sep 20;146(6):1265-76
references.pmid: 10491390
references.citation: Biochemistry. 2010 May 4;49(17):3695-702
references.pmid: 20192276
references.citation: J Neurosci. 2006 Oct 4;26(40):10243-52
references.pmid: 17021180
references.citation: Genomics. 2000 Aug 1;67(3):333-42
references.pmid: 10936054
references.citation: Biophys J. 2000 Sep;79(3):1524-9
references.pmid: 10969013
references.citation: Biochemistry. 1996 Jul 23;35(29):9392-9
references.pmid: 8755717
references.citation: J Biol Chem. 2006 Dec 8;281(49):37291-301
references.pmid: 17012748
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7518-23
references.pmid: 12032315
references.citation: J Biol Chem. 2006 Oct 20;281(42):31987-94
references.pmid: 16920704
references.citation: Science. 2001 Aug 17;293(5533):1317-20
references.pmid: 11509731
references.citation: J Cell Biol. 2003 Feb 3;160(3):409-21
references.pmid: 12566431
references.citation: Curr Opin Cell Biol. 2004 Feb;16(1):61-7
references.pmid: 15037306
references.citation: Biochem Biophys Res Commun. 2004 Jun 18;319(1):214-20
references.pmid: 15158464
references.citation: Proc Natl Acad Sci U S A. 1982 Oct;79(20):6151-5
references.pmid: 6959106
references.citation: Neuron. 2004 Jul 8;43(1):95-103
references.pmid: 15233920
references.citation: J Cell Sci. 2004 Sep 15;117(Pt 20):4797-806
references.pmid: 15340015
references.citation: Physiol Rev. 2004 Jan;84(1):1-39
references.pmid: 14715909
references.citation: Cell. 1981 Jan;23(1):175-82
references.pmid: 6260373
references.citation: J Biol Chem. 1996 Jul 19;271(29):16983-6
references.pmid: 8707782
references.citation: Biochem Biophys Res Commun. 2004 Mar 12;315(3):538-45
references.pmid: 14975734
references.citation: Nat Cell Biol. 2009 Apr;11(4):443-50
references.pmid: 19287378
references.citation: J Cell Biol. 2004 Mar 15;164(6):877-86
references.pmid: 15007063
references.citation: Biophys J. 2008 Dec 15;95(12):5706-18
references.pmid: 18936243
references.citation: J Biol Chem. 2003 Jun 13;278(24):21352-60
references.pmid: 12672820
references.citation: Cell. 1988 Mar 11;52(5):757-72
references.pmid: 2449973
references.citation: J Cell Sci. 2004 Nov 15;117(Pt 24):5825-34
references.pmid: 15522885
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: J Biochem. 1982 Sep;92(3):871-9
references.pmid: 7142124
references.citation: Curr Biol. 1998 Jul 30-Aug 13;8(16):903-13
references.pmid: 9707401
references.citation: Mol Biol Cell. 2003 Oct;14(10):4173-80
references.pmid: 14517327
references.citation: Mol Cell. 2009 Mar 13;33(5):537-45
references.pmid: 19285938
references.citation: Biochemistry. 2008 Feb 26;47(8):2485-96
references.pmid: 18229949
references.citation: Philos Trans R Soc Lond B Biol Sci. 2000 Apr 29;355(1396):419-31
references.pmid: 10836495
references.citation: J Biol Chem. 2007 Jan 5;282(1):216-31
references.pmid: 17074769
references.citation: Cell. 1996 Apr 19;85(2):149-58
references.pmid: 8612268
references.citation: J Cell Biol. 2007 Oct 22;179(2):229-38
references.pmid: 17954606
references.citation: Nat Cell Biol. 2005 Feb;7(2):148-56
references.pmid: 15654330