Differential effects of caldesmon on the intermediate conformational states of polymerizing actin.
The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.
Huang
Renjian
R
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Grabarek
Zenon
Z
Wang
Chih-Lueh Albert
CL
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D003198:Computer Simulation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011721:Pyrenes / Q000378:metabolism; D011817:Rabbits; D012984:Software; D013997:Time Factors
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D011721:Pyrenes; C030984:pyrene; D048049:Extracellular Signal-Regulated MAP Kinases