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 Gαq. 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 Gαq to Rho appeared to be inhibited by MLK3. Overexpression of MLK3 or a kinase-dead form, MLK3KR, along with a constitutively active form of Gαq, 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 Gαq, showed an increased interaction between MLK3KR and a tagged form of p63RhoGEF, and overexpression of MLK3KR decreased the carbachol-stimulated activation of Rho. When activated Gαq was coexpressed with tagged p63RhoGEF and increasing amounts of tagged MLK3, MLK3 inhibited the interaction between Gαq 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 MLK3KR 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 Gαq-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 Gαq signaling to Rho by binding to p63RhoGEF. Mol. Cell 32, 43-56 (2008). [PubMed]