Phosphorylation of tropomodulin1 contributes to the regulation of actin filament architecture in cardiac muscle

Abstract

Tropomodulin1 (Tmod1) is an actin-capping protein that plays an important role in actin filament pointed-end dynamics and length in striated muscle. No mechanisms have been identified to explain how Tmod1's functional properties are regulated. The purpose of this investigation was to explore the functional significance of the phosphorylation of Tmod1 at previously identified Thr54. Rat cardiomyocytes were assessed for phosphorylation of Tmod1 using Pro-Q Diamond staining and (32)P labeling. Green fluorescent protein-tagged phosphorylation-mimic (T54E) and phosphorylation-deficient (T54A) versions of Tmod1 were expressed in cultured cardiomyocytes, and the ability of these mutants to assemble and restrict actin lengths was observed. We report for the first time that Tmod1 is phosphorylated endogenously in cardiomyocytes, and phosphorylation at Thr54 causes a significant reduction in the ability of Tmod1 to assemble to the pointed end compared with that of the wild type (WT; 48 vs. 78%, respectively). In addition, overexpression of Tmod1-T54E restricts actin filament lengths by only ∼3%, whereas Tmod1-WT restricts the lengths significantly by ∼8%. Finally, Tmod1-T54E altered the actin filament-capping activity in polymerization assays. Taken together, our data suggest that pointed-end assembly and Tmod1's thin filament length regulatory function are regulated by its phosphorylation state.

Actin regulation by tropomodulin and tropomyosin in neuronal morphogenesis and function

“…TRPM7 phosphorylates Tmod1 (Dorovkov et al, 2008) and Tmod2 (Kostyukova, Dorovkov, unpublished) in vitro. Overexpression of a phospho-mimic Tmod1 in cardiomyocytes had weakened association with pointed ends and did not decrease thin filament length like either of the wild-type or phospho-null mutant (Bliss et al, 2014). TRPM7 is involved in actin regulation and cell death in neurons (for review see (Asrar and Aarts, 2013)).…”

“…Tmods can be phosphorylated; for example, Tmod1 was found to be phosphorylated in cardiomyocytes (Bliss et al, 2014). TRPM7 phosphorylates Tmod1 (Dorovkov et al, 2008) and Tmod2 (Kostyukova, Dorovkov, unpublished) in vitro.…”

Tropomodulin1 directly controls thin filament length in both wild-type and tropomodulin4-deficient skeletal muscle

“…Although it has been known for some time that pointed-end capping by sarcomeric Tmods is inversely related to thin filament length in cardiac myocytes as well as Drosophila indirect flight muscle (Bliss et al, 2014;Gregorio et al, 1995;Littlefield et al, 2001;Mardahl-Dumesnil and Fowler, 2001;Sussman et al, 1998), the only evidence for Tmod participation in thin filament length regulation in skeletal muscle has been indirect evidence obtained from dystrophic mouse muscles, in which m-calpain-induced proteolysis of Tmod1 is associated with thin filament elongation (Gokhin et al, 2014b). However, the utility of dystrophic muscles for studying Tmodmediated thin filament length regulation is constrained by the fact that residual unproteolyzed Tmod remains in muscle after Ca 2+ influx and m-calpain induction, and that the extent of m-calpain proteolysis is highly variable and depends on both the muscle type and the severity of the dystrophy (Gokhin et al, 2014b).…”

Overexpression of TMOD1 is associated with enhanced regional lymph node metastasis in human oral cancer

“…TMOD1-4 are 70% similar in amino acid sequence with different expression profiles (13) and contain an N-terminal unstructured domain and a C-terminal domain consisting of 5 leucine-rich repeat motifs (14,15). TMOD1-4 inhibit elongation and depolymerization of actin filaments by binding to the pointed end of the actin filament (16)(17)(18).…”