Cell Biology MLK3 as a RhoGEF Partner

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

Mixed lineage kinase 3 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that can activate multiple MAPKs, including c-Jun N-terminal kinase (JNK). However, all of MLK3's functions may not be mediated by activation of MAPK signaling. Swenson-Fields et al. report that MLK3 can interact with a Rho guanine nucleotide exchange factor (GEF), p63RhoGEF (and the short form p63RhoGEFT), to inhibit its interaction with Rho, which inhibits Rho activation by G protein-coupled receptors (GPCRs) that signal through G_q. RNA interference experiments with cultured cells showed that knockdown of MLK3 inhibited chemotaxis (transwell migration assay), increased the size of focal adhesions and the size and thickness of stress fibers, and increased the amount of activated Rho present in the cells (Rhotekin pull-down assay). Surprisingly, the cells in which MLK3 was knocked down did not show decreased MAPK signaling in response to epidermal growth factor stimulation, suggesting that MAPK signaling was not compromised in the absence of MLK3. Instead, signaling from G_q to Rho appeared to be inhibited by MLK3. Overexpression of MLK3 or a kinase-dead form, MLK3^KR, along with a constitutively active form of G_q, decreased the activity of a serum response factor (SRF) reporter gene, which was used as a measure of Rho activity. Affinity purification assays, in vitro assays, and coimmunoprecipitation assays revealed that tagged versions of MLK3 and p63RhoGEF interacted. Cells exposed to the muscarinic ligand carbachol, which stimulates a GPCR that is coupled to G_q, showed an increased interaction between MLK3^KR and a tagged form of p63RhoGEF, and overexpression of MLK3^KR decreased the carbachol-stimulated activation of Rho. When activated G_q was coexpressed with tagged p63RhoGEF and increasing amounts of tagged MLK3, MLK3 inhibited the interaction between G_q and p63RhoGEF. The interaction of MLK3 and p63RhoGEF appeared to be regulated by the phosphorylation state of MLK3. Phosphomimetic mutants (residues Thr²⁷⁷ and Ser²⁸¹, which are activating phosphorylation events that occur in response to GPCR signaling) of MLK3^KR exhibited increased interaction with p63RhoGEF. In contrast, feedback phosphorylation by JNK appears to inhibit the interaction between MLK3 and p63RhoGEF. Thus, MLK3 appears to have a kinase-independent role in controlling Rho activation by G_q-coupled GPCRs.

K. I. Swenson-Fields, J. C. Sandquist, J. Rossol-Allison, I. C. Blat, K. Wennerberg, K. Burridge, A. R. Means, MLK3 limits activated G_q signaling to Rho by binding to p63RhoGEF. Mol. Cell 32, 43-56 (2008). [PubMed]

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In Science Signaling

EDITORS' CHOICE
SIGNALING SPECIFICITY
Directing Rho Signals

(15 March 2005)
Sci. STKE 2005 (275), tw102. [DOI: 10.1126/stke.2752005tw102]
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