Modes of caldesmon binding to actin

Sites of caldesmon contact and modulation of interactions by phosphorylation

Darren Brian Foster, Renjian Huang, Victoria Hatch, Roger Craig, Philip Graceffa, William Lehman, C. L Albert Wang

Research output: Contribution to journalArticle

Abstract

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys595, away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

Original languageEnglish (US)
Pages (from-to)53387-53394
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number51
DOIs
StatePublished - Dec 17 2004

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Calmodulin-Binding Proteins
Phosphorylation
Actins
Modulation
Extracellular Signal-Regulated MAP Kinases
Actin Cytoskeleton
Muscle
Actomyosin
Myosins
Depolymerization
Smooth Muscle Myocytes
Smooth Muscle
Electron Microscopy
Polymers
Protein Isoforms
Electron microscopy
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Modes of caldesmon binding to actin : Sites of caldesmon contact and modulation of interactions by phosphorylation. / Foster, Darren Brian; Huang, Renjian; Hatch, Victoria; Craig, Roger; Graceffa, Philip; Lehman, William; Wang, C. L Albert.

In: Journal of Biological Chemistry, Vol. 279, No. 51, 17.12.2004, p. 53387-53394.

Research output: Contribution to journalArticle

Foster, Darren Brian ; Huang, Renjian ; Hatch, Victoria ; Craig, Roger ; Graceffa, Philip ; Lehman, William ; Wang, C. L Albert. / Modes of caldesmon binding to actin : Sites of caldesmon contact and modulation of interactions by phosphorylation. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 51. pp. 53387-53394.
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