Regulatory Mechanisms of Myosin I in Dictyostelium discoideum

Abstract

The class I myosins are an ubiquitous family of non-filamentous, single-headed actin-binding motor proteins. The objective of this study was to identify the light chain composition of the short-tailed Dictyostelium class I myosins, MyoIA and MyoIE. Flag-tagged MyoIA head-neck and MyoIE head-neck constructs were generated and expressed in Dicyostelium discoidem. The MyoIA and MyoIE head-neck constructs both co-purified with a 17-kDa protein that reacted with an anti-calmodulin antibody and exhibited a mobility shift on SDS gels in the presence of calcium. Mass spectrometry analysis confirmed that the light chain bound to MyoIA and MyoIE was calmodulin. The finding that the short-tailed class I Dictyostelium myosins use the generic calcium-binding protein calmodulin as a light chain contrasts with previous work showing that the long-tailed Dictyostelium class I myosins MyoIB, MyoIC, and MyoID each bind a unique, specialized light chain called MlcB, MlcC, and MlcD, respectively. Despite having a calmodulin light chain, calcium did not affect the actin-activated Mg-ATPase activities of MyoIA or MyoIE. The p21-activated kinases (PAKs) are serine-threonine protein kinases that are activated by the small GTPases Cdc42 and Rac. PAKs phosphorylate a site in the motor domain of Dictyostelium class I myosins that is required for myosin activity. Studies were carried out to determine whether Dictyostelium RacB, which is known to bind to and activate Dictyostelium PAKs, promotes the phosphorylation of MyoID in vivo. A vector that expresses a constitutively active RacB under the control of a doxycycline-inducible promoter was created and transformed into Dictyostelium cells. Immunostaining demonstrated that the constitutively active RacB increased actin filament formation in AX3 cells by ~3-fold but by only ~1.5-fold in PakB-null cells. A rabbit polyclonal antibody against the MyoID tail was made. An anti-phospho antibody raised against a phosphorylated peptide corresponding to the MyoID TEDS site was tested and found to specifically recognize purified phosphorylation MyoIA and MyoID. The anti-phospho antibody did not detect phosphorylated MyoIA or MyoID in crude Dictyostelium cell extracts or in immunoprecipitates prepared using the anti-MyoID antibody. Further work is needed to improve the specificity of the anti-phospho MyoID antibody.