Abstract
We showed previously that phosphorylation of Tyr(53), or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S., Hong, M. S., Levine, R. L., and Korn, E. D. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13694-13699; Liu, X., Shu, S., Hong, M. S., Yu, B., and Korn, E. D. (2010) J. Biol. Chem. 285, 9729-9739). We now show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, although individual amoebae chemotax normally. We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMP-induced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization. Trafficking of vesicles containing adenylyl cyclase A to the rear of the cell and secretion of the ACA vesicles are also inhibited. The actin cytoskeleton of cells expressing Y53A-actin is characterized by numerous short filaments, and bundled and aggregated filaments similar to the structures formed by copolymerization of purified Y53A-actin and wild-type actin in vitro. This disorganized actin cytoskeleton may be responsible for the inhibition of intracellular and intercellular cAMP signaling in cells expressing F-Y53A-actin.
Highlights
Dictyostelium discoideum grows as single-cell amoebae on yeast, bacteria, or soluble medium with a generation time in liquid medium of about 8 –12 h
We show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, individual amoebae chemotax normally
We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMPinduced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization
Summary
A FLAG sequence (DYKDDDDK) was added to the N terminus of the mutant actin (F-Y/A), and to WT-actin (F-Y/Y) as a control for possible effects of the FLAG tag. Actin cDNAs were cloned onto a pTIKL vector, which has an actin 15 promoter and G-418 resistance [23]. Expression plasmids were introduced into Dictyostelium AX3 cells using a gene pulser (Bio-Rad) [24]. For coexpression of actin and ACA in acaϪ cells, the actin and promoter sequences amplified by PCR from pTIKL F-Y/Yactin and pTIKL F-Y/A-actin were subcloned into pCV5 using restriction enzymes XbaI and BglII and the sequences were confirmed. Equal amounts of pCV5 ACA-YFP [25] and pCV5 actin were mixed and transfected into acaϪ cells [25] by electroporation
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