Editors' ChoiceSystems Biology

Switching Partners by Phosphorylation

Sci. Signal.  22 Jul 2014:
Vol. 7, Issue 335, pp. ec199
DOI: 10.1126/scisignal.2005715

Cell proliferation is stimulated by the mitogen-activated protein kinase (MAPK) pathway and suppressed by the Hippo pathway. Romano et al. found that crosstalk between the MAPK and Hippo pathways is mediated by phosphorylation-regulated competition for interaction with the kinase RAF-1. Serum stimulation decreased the interaction between RAF-1 and the Hippo pathway kinase MST2, resulting in inhibition of MST2, and increased that between RAF-1 and the MAPK MEK, resulting in activation of MEK. RAF-1 competes with RAS association domain–containing protein 1 (RASSF1A) for binding to MST2. The activity of MST2, MEK, and MEK’s substrate extracellular signal–regulated protein kinase (ERK) was increased in MCF7 cells by overexpression of RASSF1A (MCF7 cells lack RASSF1A) and decreased in HeLa cells by knockdown of endogenous RASSF1A. A mathematical model indicated that posttranslational modifications altered protein binding affinity. In unstimulated MCF7 cells, RAF-1 interacted with MST2 and was phosphorylated at its inhibitory site, Ser259. In contrast, serum stimulation, expression of RASSF1A, or expression of a nonphosphorylatable form of RAF1 (S259A) decreased the phosphorylation of Ser259 on RAF-1 and increased phosphorylation of the activating site, Ser338. Phosphorylation of Ser259, which maintains the RAF-1–MST2 interaction, was mediated in vitro by the Hippo pathway kinase LATS1 (a substrate of MST2), and both the phosphorylation and the interaction were reduced by LATS1 RNA interference in HeLa cells and in LATS1–/– mouse embryonic fibroblasts (MEFs). The RAF-1–MEK interaction was increased in LATS1–/– MEFs. The switch to a RAF-1–MEK interaction by overexpression of the S259A RAF-1 mutant accelerated the proliferation of HeLa cells and induced clonogenic growth in NIH3T3 MEFs, which were further enhanced by depletion of MST2. In addition, zebrafish expressing the S259A RAF-1 mutant had enlarged hearts. Together the findings suggest that the balance between the Hippo and MAPK pathways is regulated by phosphorylation-sensitive binding to RAF-1.

D. Romano, L. K. Nguyen, D. Matallanas, M. Halasz, C. Doherty, B. N. Kholodenko, W. Kolch, Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling. Nat. Cell Biol. 16, 673–684 (2014). [PubMed]