Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration.
Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.
Eppinga
Robbin D
RD
Department of Biological Sciences, University of Iowa, Iowa City 52242-1324, USA.
Li
Yan
Y
Lin
Jenny L-C
JL
Mak
Alan S
AS
Lin
Jim J-C
JJ
D000409:Alanine / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D006224:Cricetinae; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011554:Pseudopodia / Q000502:physiology*; D022502:Stress Fibers / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology; D054462:p21-Activated Kinases
D002148:Calmodulin-Binding Proteins; D018698:Glutamic Acid; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D000409:Alanine