Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase

Xiang Qun Hu, Namita Singh, Debabrata Mukhopadhyay, Hamid I. Akbarali

Research output: Contribution to journalArticle

105 Citations (Scopus)

Abstract

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of ~60 and 125 kDa were observed in immunoblots using an anti- phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co- immunoprecipitated with c-Src, and the phosphorylation of the c-Src-FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c- Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti- Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The α(1C) subunit of the L-type Ca2+ channel co- immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF- induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.

Original languageEnglish (US)
Pages (from-to)5337-5342
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number9
DOIs
StatePublished - Feb 27 1998
Externally publishedYes

Fingerprint

Focal Adhesion Protein-Tyrosine Kinases
Smooth Muscle Myocytes
Muscle
Cells
Modulation
Rabbits
Antibodies
Electric potential
Dialysis
Anti-Idiotypic Antibodies
Phosphotyrosine
Src peptide
Platelet-Derived Growth Factor
Immunoblotting
Mitogen-Activated Protein Kinase Kinases
Muscle Contraction
Mitogen-Activated Protein Kinases
Protein-Tyrosine Kinases
Smooth Muscle
Tyrosine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. / Hu, Xiang Qun; Singh, Namita; Mukhopadhyay, Debabrata; Akbarali, Hamid I.

In: Journal of Biological Chemistry, Vol. 273, No. 9, 27.02.1998, p. 5337-5342.

Research output: Contribution to journalArticle

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AU - Singh, Namita

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N2 - There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of ~60 and 125 kDa were observed in immunoblots using an anti- phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co- immunoprecipitated with c-Src, and the phosphorylation of the c-Src-FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c- Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti- Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The α(1C) subunit of the L-type Ca2+ channel co- immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF- induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.

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