Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

Abstract

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division of Xenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

“…phosphorylation at Ser/Thr residues in the C-terminal region, leading to a weakened affinity toward the actin filament. Furthermore, prevention of l-CaD phosphorylation at all potential cdc2 sites by mutagenesis indeed slows down, although does not stop, the progression of cell cycle [5,51]. Nevertheless, several important gaps remain: Direct evidence is missing for CaD phosphorylation in individual cells at various cell cycle stages, since in these studies anti-CaD antibodies were used in microscopic imaging, and phosphorylation of l-CaD was inferred only by subsequent analysis of 32 P incorporation of cells arrested at the corresponding stages [48][49][50].…”

“…Such phenomena are consistent with the idea that unphosphorylatable l-CaD remains bound to actin filaments even when pertinent kinases are activated, rendering the overly stabilized actin cytoskeleton more difficult to remodel itself. However, like in the case when all seven cdc2 kinase sites were mutated mitosis was only delayed, yet not stopped [5,7], the mutation at Erk sites fails to prevent the cell from eventually changing its shape. Apparently, there are other sites in this region that can be modified by alternative signaling pathways.…”

“…In the smooth muscle system, CaD stabilizes the filamentous organization and inhibits actomyosin interactions [1]. In non-muscle cells CaD is involved in cell migration [2]; it also inhibits Arp2/3 assembly [3,4] and interferes with cell cycle progression [5][6][7]. Binding of CaD to actin is modulated by Ca 2+ /calmodulin (CaM) as well as phosphorylation through kinases such as MAPK, Pak and p34 cdc2 ( Figure 1).…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…Prevention of CaD phosphorylation at all potential cdc2 sites by mutagenesis indeed slows down progression through the cell cycle. 156,170 More direct evidence for CaD phosphorylation in individual cells at various cell cycle stages was obtained by immuno-staining studies. Anti-phosphopeptide antibodies revealed a dynamic change of CaD phosphorylation throughout the cell cycle progression, in a manner that is reciprocal to the change of actin stress fibers.…”

“…When the cdc2-sites or the ERK-sites were blocked by mutagenesis, cell division and detachment were slowed down, but not stopped, 171,178 indicating that there must be alternative pathways that cells can adopt to circumvent the impediment. Indeed, CaD is also known to be phosphorylated by the p21-activated protein kinase (PAK).…”