Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-δ2

Paul J. Pfleiderer, Katherine Kun Lu, Michael T. Crow, Rebecca S. Keller, Harold A. Singer

Research output: Contribution to journalArticle

Abstract

Previous studies demonstrated a requirement for multifunctional Ca 2+/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (δ2 or δc) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKIIδ2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ 2 phosphorylated substrate in vitro without added Ca 2+/calmodulin and in the intact cell without added Ca 2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number6 55-6
DOIs
StatePublished - Jun 2004

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases
Vascular Smooth Muscle
Smooth Muscle Myocytes
Cell Movement
Muscle
Cells
Modulation
Phosphotransferases
Phosphorylation
Substrates
Protein Isoforms
Chemical activation
Ionomycin
Calmodulin
Adenoviridae
Pharmacology
Peptides
In Vitro Techniques

Keywords

  • Adenovirus
  • Autophosphorylation
  • Calcium/calmodulin-dependent protein kinase II
  • Cell migration
  • Chemotaxis
  • Platelet-derived growth factor

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-δ2. / Pfleiderer, Paul J.; Lu, Katherine Kun; Crow, Michael T.; Keller, Rebecca S.; Singer, Harold A.

In: American Journal of Physiology - Cell Physiology, Vol. 286, No. 6 55-6, 06.2004.

Research output: Contribution to journalArticle

Pfleiderer, Paul J. ; Lu, Katherine Kun ; Crow, Michael T. ; Keller, Rebecca S. ; Singer, Harold A. / Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-δ2. In: American Journal of Physiology - Cell Physiology. 2004 ; Vol. 286, No. 6 55-6.
@article{e6789f84d93842659c68a85e93002c80,
title = "Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-δ2",
abstract = "Previous studies demonstrated a requirement for multifunctional Ca 2+/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (δ2 or δc) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKIIδ2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ 2 phosphorylated substrate in vitro without added Ca 2+/calmodulin and in the intact cell without added Ca 2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.",
keywords = "Adenovirus, Autophosphorylation, Calcium/calmodulin-dependent protein kinase II, Cell migration, Chemotaxis, Platelet-derived growth factor",
author = "Pfleiderer, {Paul J.} and Lu, {Katherine Kun} and Crow, {Michael T.} and Keller, {Rebecca S.} and Singer, {Harold A.}",
year = "2004",
month = "6",
doi = "10.1152/ajpcell.00536.2003",
language = "English (US)",
volume = "286",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "6 55-6",

}

TY - JOUR

T1 - Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-δ2

AU - Pfleiderer, Paul J.

AU - Lu, Katherine Kun

AU - Crow, Michael T.

AU - Keller, Rebecca S.

AU - Singer, Harold A.

PY - 2004/6

Y1 - 2004/6

N2 - Previous studies demonstrated a requirement for multifunctional Ca 2+/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (δ2 or δc) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKIIδ2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ 2 phosphorylated substrate in vitro without added Ca 2+/calmodulin and in the intact cell without added Ca 2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.

AB - Previous studies demonstrated a requirement for multifunctional Ca 2+/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (δ2 or δc) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKIIδ2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ 2 phosphorylated substrate in vitro without added Ca 2+/calmodulin and in the intact cell without added Ca 2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.

KW - Adenovirus

KW - Autophosphorylation

KW - Calcium/calmodulin-dependent protein kinase II

KW - Cell migration

KW - Chemotaxis

KW - Platelet-derived growth factor

UR - http://www.scopus.com/inward/record.url?scp=2442473978&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2442473978&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.00536.2003

DO - 10.1152/ajpcell.00536.2003

M3 - Article

C2 - 14761894

AN - SCOPUS:2442473978

VL - 286

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 6 55-6

ER -