Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

Abstract

Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

Phospho‐regulation of intrinsically disordered proteins for actin assembly and endocytosis

“…Budding yeast has only one coronin, Crn1, which is most similar to type I coronin in vertebrates. Crn1 was also highly phosphorylated by the Clb2–Cdk1 complex , and future functional studies will hopefully yield further insights into the mechanisms underlying the Crn1 phospho‐regulation.…”

“…IDPs play essential regulatory roles as network hubs in multiple biological processes, including the progression of cell cycle, endocytosis, the formation of cytoskeleton network, and the formation of nonmembranous organelles [55,61,[64][65][66][67]. In some instances, phosphorylation appears to promote a transition to an ordered state while other examples appear to show the opposite effect.…”

“…This residue lies within a flexible loop between the adjacent EF-hand domain and actin-binding domain. Conformational simulations suggest that this Cdk1mediated phosphorylation of Sac6 alters the conformational flexibility of the loop and thus regulates the binding affinity between Sac6 and actin filaments [66]. Indeed, other examples of phosphorylation functioning as a regulatory mechanism for actin cross-linking or bundling proteins have been identified in proteins such as filamin, plastin/fimbrin and villin [154][155][156][157][158][159][160].…”

An Effective Recombinant Protein Expression and Purification System in Saccharomyces cerevisiae

“…The pYeast Pro vector (pGAL-ORF-3×StreptagII-9×His) is modified from an E. coli cloning vector pBR322; the protein is expressed under the galactose promotor (Miao et al, 2016;Peng et al, 2015;St-Pierre et al, 2009 2. Transform the ligated plasmids into competent E. coli (see Seidman et al, 1997), and plate on LB plates containing 100 µg/ml ampicillin.…”

“…The genotype of the YMY1032 yeast strain is: MATa ade2 leu2 his3 trp1 ura3 lys2::pGAL1:GAL4::LYS2 pep4::HIS3 bar1::hisG (Miao et al, 2016;Peng et al, 2015;St-Pierre et al, 2009).…”