The Tumor Suppressor RASSF1A Prevents Dephosphorylation of the Mammalian STE20-like Kinases MST1 and MST2

Abstract

The RASSF1A tumor suppressor protein interacts with the pro-apoptotic mammalian STE20-like kinases MST1 and MST2 and induces their autophosphorylation and activation, but the mechanism of how RASSF1A activates MST1/2 is unclear. Okadaic acid treatment and PP2A knockdown promoted MST1/2 phosphorylation. Data from dephosphorylation assays and reduced activation of MST1/2 seen after RASSF1A depletion suggest that dephosphorylation of MST1/2 on Thr-183 and Thr-180 by PP2A is prevented by RASSF1A, shifting the balance of MST1/2 to the activated autophosphorylated form. In addition to preventing dephosphorylation, RASSF1A also stabilized the MST2 protein. Through binding to MST1/2, RASSF1A supports maintenance of MST1/2 phosphorylation, promoting an active state of the MST kinases and favoring induction of apoptosis. This is one of the first examples of a tumor suppressor acting as an inhibitor of a specific dephosphorylation pathway.The RASSF1A (Ras association domain family 1 isoform A) gene is localized on chromosome 3p21.3, in an area that likely harbors at least one important tumor suppressor gene (1, 2). RASSF1A is frequently silenced by promoter hypermethylation in many human tumors (3, 4). Rassf1a-targeted mice are prone to spontaneous and carcinogen-induced tumorigenesis (5, 6). The RASSF1A protein is involved in several growth regulatory and pro-apoptotic pathways. Recent work has shown that RASSF1A associates with components of the mammalian Hippo signaling network (7), an emerging pathway implicated in organ size control, restriction of cell proliferation, apoptosis, and tumor suppression (8 -10). RASSF1A interacts with the mammalian kinases MST1 and MST2, the orthologs of the Drosophila Hippo kinase, and activates MST1 and MST2 by promoting their autophosphorylation and phosphorylation of the downstream LATS1 kinase (7).The mammalian Sterile-20-like kinases MST1 (also known as STK4 and KRS2) and MST2 (also known as STK3 and KRS1) belong to the class II germinal center (Ser/Thr protein) kinases (11). MST1 and MST2 have recently been implicated as important tumor suppressors (12)(13)(14), suggesting that the RASSF-MST complexes may represent intriguing tumor-suppressing modules. Besides their potential role in promoting apoptosis through the Hippo pathway, MST1 and MST2 are implicated in several other pro-apoptotic processes. During induction of apoptosis, MST1 and MST2 can be activated, leading to phosphorylation of histone H2B and nuclear DNA fragmentation (15). In addition, the activation of JNK (Jun N-terminal kinase) signaling (16, 17) and phosphorylation of FOXO3 transcription factors (18) have also been associated with MST-induced apoptosis.The MST1 and MST2 kinases are regulated by several mechanisms, including phosphorylation, caspase cleavage, dimerization, and cofactors (19). They are activated in response to staurosporine (STS), 2 a potent protein kinase C inhibitor and apoptosis inducer, or the protein phosphatase inhibitor okadaic acid (20). Several other pro-apoptotic stimuli and stresses hav...

3,3′-Diindolylmethane suppresses the growth of gastric cancer cells via activation of the Hippo signaling pathway

“…Targeted disruption of the RASSF1 gene in mice resulted in spontaneous and carcinogen-induced tumorigenesis (46), indicating that RASSF1 serves as an important tumor-suppressor. Recent research has shown that Hippo signal transduction pathway activity is regulated by RASSF1, an emerging pathway implicated in the control of cell growth, apoptosis, and tumor-suppression (20,41,47). In addition, several studies have shown that RASSF1 interacts with the mammalian Mst1/2, and promotes phosphorylation of Mst1/2 and their downstream gene products (48,49), suggesting that RASSF1-Mst1/2 may have intriguing tumor-suppressing characteristics.…”

“…RASSF1 has been reported to interact with the mammalian kinases, Mst1, and Mst2 and activate them by promoting their autophosphorylation and subsequent phosphorylation of the downstream LATS1 kinase (41). In addition, several studies have revealed that Hippo signaling is tightly regulated by protein-protein interactions (14).…”

Mechanisms of Hippo pathway regulation

“…It has not been shown whether SAV1 directly affects MST1/2 kinase activity. The functional role of RASSFs in MST1/2 regulation can be either positive or negative and may depend on the status of MST1/2 (Khokhlatchev et al 2002;Praskova et al 2004;Guo et al 2007Guo et al , 2011. Nevertheless, it is evident that RASSFs disrupt MST1/2 dimerization and prevent their autophosphorylation.…”

“…Nevertheless, it is evident that RASSFs disrupt MST1/2 dimerization and prevent their autophosphorylation. However, interaction of RASSFs with already activated MST1/2 may prevent MST1/2 dephosphorylation and therefore sustain MST1/2 kinase activity (Guo et al 2011;Ni et al 2013).…”

Hippo signalling in the G2/M cell cycle phase: Lessons learned from the yeast MEN and SIN pathways

“…In human cells, RASSF1A signals through MST/LATS1 to induce apoptosis [105][106][107], and can also trigger apoptosis by MST1/NDR signalling [108]. Part of the mode of action appears to be that RASSF1A protects MST1/2 from dephosphorylation by PP2A to induce apoptosis [109]. Whatever the case, RASSF1A has been repeatedly reported as an activator of MST1/2 signalling [105][106][107][108]110].…”