Activation of the Mitogen-activated Protein Kinase ERK2 by the Chemoattractant Folic Acid in Dictyostelium

Abstract

In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity. Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled G␣ subunit G␣4 are maximally expressed. During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE. Interestingly, this activation is not elicited in the absence of G␤ subunits, in contrast to the response to cAMP. This response also requires the G␣4 subunit known to be required for other folic acidmediated responses (Hadwiger, J., Lee, S., and Firtel, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10566 -10570). Furthermore, we show that the activation of ERK2 by cAMP is independent of the G␣4 subunit, while the activation of ERK2 by folate is independent of G␣2. Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G proteindependent and -independent pathways. MAP 1 kinase cascades are used by eukaryotes to couple extracellular signals to diverse intracellular regulatory pathways. The diversity of these pathways is great and includes growth factor responses in mammalian cells, cell-type differentiation in Drosophila and Caenorhabditis elegans, mating and growth responses in yeast, and stress-mediated responses in yeast and mammalian cells. These cascades are mediated by various classes of G protein-and tyrosine kinase-coupled receptors (5-13). In Dictyostelium, three presumably independent MAP kinase cascades play essential regulatory roles during both growth and multicellular development. The MAP kinase kinase (MEK) DdMEK1 is required for chemotaxis toward cAMP during aggregation; ddmek1 null cells form very small aggregates but continue to differentiate into normally proportioned, but very small, fruiting bodies (14). DdMEK1 is specifically required at the time of cAMP stimulation for the activation of guanylyl cyclase and the production of cGMP, the second messenger for chemotaxis in these cells. Two MAP kinases, ERK1 and ERK2, have been identified and their functions characterized. ERK1 is required for vegetative growth and is thought to play roles during multicellular development (15), while ERK2 is required for the activation of adenylyl cyclase by the chemoattractant cAMP during aggregation, prespore-specific gene expression, and morphogenesis (2, 16). erk2 null cells are aggregation-deficient due to the inability to activate adenylyl cyclase and relay the cAMP signal but show normal activation of guanylyl cyclase, which couples to chemotaxis (17). erk2 null cells expressing a temperature-sensitive ERK2 show abnormal morphogenesis during the multicellular stages, as well as the inability to induce prespore gene expression when shifted to a nonpermissive temperature. ERK2 activity is induce ϳ...

Constitutively Active G Protein-coupled Receptor Mutants BlockDictyosteliumDevelopment

“…To determine whether the dominantnegative receptors also impair G protein-independent responses mediated by cAR1, we examined the stimulation of mitogen-activated protein kinase (MAPK) activity by cAMP and, for comparison, folate. Both ligands activate primarily the ERK2 isoform of MAPK in a transient manner with peak activation occurring after ϳ50 s of stimulation (Maeda et al, 1996;Maeda and Firtel, 1997). As shown by immunoblotting with antibodies specific for activated phospho-MAPK (Figure 6C), robust activation was obtained with folate for cAR1 cells as well as DN3 and DN4 cells.…”

Paxillin is required for cell-substrate adhesion, cell sorting and slug migration duringDictyosteliumdevelopment

“…Mutation of these sites also did not influence the rate of assembly in the characteristic PaxB spots although they could affect the interaction with other proteins not rate limiting in the assembly of the complexes. There are however a number of other protein kinases especially of the MAP kinase family that have been implicated in chemotaxis (Gaskins et al, 1996;Kosaka et al, 1998;Kosaka and Pears, 1997;Maeda and Firtel, 1997;Wang et al, 1998). A first step towards establishing whether phosphorylation plays a role in paxillin regulation will be to determine, which sites are phosphorylated in vivo after the cells contact different substrates and after stimulation with chemo-attractants such as cAMP.…”

Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

“…Although vegetative cells are amoeboid-shaped and unpolarized, they are quite capable of migrating directionally in a FA gradient (Bernstein et al, 1981;de Wit and Rinke de Wit, 1986;Devreotes, 1983;Hadwiger and Srinivasan, 1999;Jowhar et al, 2010;Kesbeke et al, 1990;Kortholt et al, 2011;Maeda and Firtel, 1997;Pan et al, 1972;van Haastert et al, 1982). On the other hand, cells that have been starved undergo developmental changes that result in a distinct polarized morphology.…”