Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

Abstract

We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKCmediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained α-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.

Caldesmon and the Regulation of Cytoskeletal Functions

“…Such an effect should be readily testable by biochemical experiments. Another notable finding in these studies is that ERK-phosphorylated CaD (although not so clear about PAK-phosphorylated CaD) is localized at the ring of podosomes 186 where Tm5a/5b was found in the osteoclasts. 58 Whether such colocalization suggests interactions or common targets is worthy of further investigation.…”

“…Interestingly, l-CaD is present in such podosomes 185 and is phosphorylated at ERK sites. 186 By overexpressing a phosphorylation-deficient CaD-GFP mutant it was found that, although both the size and the lifetime of the podosomes are modulated by ERK-mediated CaD phosphorylation, the modes of ERK and CaD in their modulation of the formation and dynamics of podosomes are different. 186 This suggests involvement of other players in addition to ERK.…”

“…186 By overexpressing a phosphorylation-deficient CaD-GFP mutant it was found that, although both the size and the lifetime of the podosomes are modulated by ERK-mediated CaD phosphorylation, the modes of ERK and CaD in their modulation of the formation and dynamics of podosomes are different. 186 This suggests involvement of other players in addition to ERK. It turned out that the formation of podosomes was more tightly correlated with PAK-mediated phosphorylation of CaD.…”

Mechanism of Flavonoids Action in Smooth Muscle Relaxation

“…The mechanism by which phorbol ester activates MEK/ ERK and causes vascular contraction has been established. [121] On the other hand, previous studies that inspected the mechanisms underlying arterial contractions induced by fluoride or thromboxane A2 have reported variable findings with regard to the contraction triggered by…”

Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

“…There is a subset of proteins that are thought to have a role in regulating podosome half-life, and that may be important regulators of podosome structure and function. Recently it has been shown that inhibition of Erk1/2 phosphorylation leads to increased podosome lifetime in PDBu-stimulated A7R5 cells (Gu et al, 2007). Through detailed mathematical modeling, it has been shown that increased podosome lifetime was mainly affected by the decreased rate constant for podosome disassembly, which indicated that Erk1/2 phosphorylation is involved in facilitating podosome disassembly.…”