Caldesmon is a cytoskeletal target for PKC in endothelium.
We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise.
Bogatcheva
Natalia V
NV
Department of Medicine, Section of Pulmonary and Critical Care, The University of Chicago Center for Integrative Science, 929 E. 57th Street, Chicago, IL 60637, USA.
Birukova
Anna
A
Borbiev
Talaibek
T
Kolosova
Irina
I
Liu
Feng
F
Garcia
Joe G N
JG
Verin
Alexander D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003598:Cytoskeletal Proteins / Q000378:metabolism; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate