Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

Abstract

On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-␤-actin and profilin-␣-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7°rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.actin phosphorylation ͉ profilin-actin structure ͉ pY53-actin structure ͉ Dictyostelium discoideum actin ͉ gelsolin-actin structure

A novel interplay between oncogenic PFTK1 protein kinase and tumor suppressor TAGLN2 in the control of liver cancer cell motility

“…As PFTK1 is a serine/ threonine protein kinase, it is probable that ACTB tyrosine phosphorylation has resulted from the indirect effects of the PFTK1 cascade, whereas TAGLN serine phosphorylation is likely consequential to a direct kinase activity. Nevertheless, it is worth noting that studies have established a close relation between actin phosphorylation and cytoskeleton remodeling (Rush et al, 2005;Baek et al, 2008). Specifically, ACTB tyrosine phosphorylation has been shown to induce considerable actin re-organization and cytoskeletal rearrangement (Rush et al, 2005).…”

“…Specifically, ACTB tyrosine phosphorylation has been shown to induce considerable actin re-organization and cytoskeletal rearrangement (Rush et al, 2005). In fact, tyrosine phosphorylation of actin can protect its D-loop structure from protease cleavage, resultant in a more stable conformation of the actin D-loop than the unphosphorylated form (Baek et al, 2008). Conformational stability of the D-loop holds much importance in conferring cell motile properties, which if unstable can result in a weakened contractile force and much reduced gliding velocity (Kubota et al, 2009).…”

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

“…As a consequence of the conformational change in the D-loop, phosphorylation of Tyr-53 reduces the rate of subtilisin cleavage of the D-loop (10), reduces the affinity of monomeric actin for pancreatic DNase I (14), and reduces the rate of nucleotide exchange in monomeric actin (14). Phosphorylation of Tyr-53 also affects actin polymerization (10).…”

“…Briefly summarized, the crystal structure of pY53-actin shows the formation of hydrogen bonds between the phosphate group and residues Gly-48, Gln-49, and Lys-61 (14), thus partially stabilizing the D-loop, which is fully disordered in unphosphorylated actin, allowing the resolution of D-loop residues Gly-42, Met-47, Gly-48, and Gln-49.…”

“…To understand the consequences of Tyr-53 phosphorylation at the molecular level, we had previously determined some of the biochemical and biophysical properties and the atomic structure of pY53-actin isolated from Dictyostelium (10,14). Briefly summarized, the crystal structure of pY53-actin shows the formation of hydrogen bonds between the phosphate group and residues Gly-48, Gln-49, and Lys-61 (14), thus partially stabilizing the D-loop, which is fully disordered in unphosphorylated actin, allowing the resolution of D-loop residues Gly-42, Met-47, Gly-48, and Gln-49.…”