title: h3/Acidic calponin: an actin-binding protein that controls extracellular signal-regulated kinase 1/2 activity in nonmuscle cells.
abstract: Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments. We show that the actin-binding protein h3/acidic calponin associates with stress fibers in the absence of stimulation but is targeted to the cell cortex and podosome-like structures after stimulation with a phorbol ester, phorbol-12,13-dibutyrate (PDBu). By coimmunoprecipitation and colocalization, we show that extracellular signal-regulated kinase (ERK)1/2 and protein kinase C (PKC)alpha constitutively associate with h3/acidic calponin and are cotargeted with h3/acidic calponin in the presence of PDBu. This targeting can be blocked by a PKC inhibitor but does not require phosphorylation of h3/acidic calponin at the PKC sites S175 or T184. Knockdown of h3/acidic calponin results in a loss of PDBu-mediated ERK1/2 targeting, whereas PKCalpha targeting is unaffected. Caldesmon is an actin-binding protein that regulates actomyosin interactions and is a known substrate of ERK1/2. Both ERK1/2 activity and nonmuscle l-caldesmon phosphorylation are blocked by h3/acidic calponin knockdown. Furthermore, h3/acidic calponin knockdown inhibits REF52.2 migration in an in vitro wound healing assay. Our findings are consistent with a model whereby h3/acidic calponin controls fibroblast migration by regulation of ERK1/2-mediated l-caldesmon phosphorylation.
authors.affiliation: Department of Health Sciences and Whitaker Imaging Facility, Biomedical Engineering, Boston University, Boston, MA 02215, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Appel
authors.forename: Philip G
authors.initials: PG
authors.lastname: Allen
authors.forename: Susanne
authors.initials: S
authors.lastname: Vetterkind
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Jin
authors.forename: Kathleen G
authors.initials: KG
authors.lastname: Morgan
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D055785:Gene Knockdown Techniques; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000201:enzymology; D041681:NIH 3T3 Cells; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D014945:Wound Healing / Q000187:drug effects; D000096985:Calponins
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015240:Phorbol 12,13-Dibutyrate; D010768:Phosphoserine; D010769:Phosphothreonine; D051571:Protein Kinase C-alpha; D047428:Protein Kinase Inhibitors; D000096985:Calponins