Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase

Abstract

The tumour suppressor protein merlin (encoded by the neurofibromatosis type 2 gene NF2) is an important regulator of proliferation in many cell and tissue types [1][2][3][4] . Merlin is activated by dephosphorylation at serine 518 (S518), which occurs on serum withdrawal or on cell-cell or cell-matrix contact 5,6 . However, the relevant phosphatase that activates merlin's tumour suppressor function is unknown. Here we identify this enzyme as the myosin phosphatase (MYPT-1-PP1d). The cellular MYPT-1-PP1d-specific inhibitor CPI-17 causes a loss of merlin function characterized by merlin phosphorylation, Ras activation and transformation. Constitutively active merlin (S518A) reverses CPI-17-induced transformation, showing that merlin is the decisive substrate of MYPT-1-PP1d in tumour suppression. In addition we show that CPI-17 levels are raised in several human tumour cell lines and that the downregulation of CPI-17 induces merlin dephosphorylation, inhibits Ras activation and abolishes the transformed phenotype. MYPT-1-PP1d and its substrate merlin are part of a previously undescribed tumour suppressor cascade that can be hindered in two ways, by mutation of the NF2 gene and by upregulation of the oncoprotein CPI-17.We tested whether carboxy-terminal merlin (amino-acid residues 299-595; C-merlin) carrying the decisive phosphorylation site S518 (ref. 6) could be dephosphorylated and could therefore be used as a substrate in vitro. C-merlin expressed in rat schwannoma RT4 cells under a doxycycline-inducible promoter (RT4tet-C-merlin) 5 migrated as a distinct doublet. The species with decreased mobility was phosphorylated and converted by treatment with calf intestinal phosphatase to a faster-migrating dephosphorylated band (Fig. 1a) with the same mobility as the non-phosphorylatable S518A mutant protein (in which S518 has been replaced with alanine, in contrast with the pseudo-phosphorylated S518D mutant protein containing an aspartic residue; Fig. 1d). The dephosphorylation of C-merlin was regulated by the same cues as that of full-length merlin 5 , for example, high cell density (Fig. 1b), the addition of high-molecular-mass hyaluronan (HA) 5 or serum withdrawal (as shown in Fig. 1c by the change in the ratio of the upper to the lower band). Because dephosphorylation took less than 5 min after hyaluronan addition or serum withdrawal, we assumed that the activation of a phosphatase was involved rather than a change in stability of the phosphorylated band.To search for the phosphatase that activates merlin's tumour suppressor function, we tested several phosphatase inhibitors for their effects on merlin dephosphorylation. Merlin dephosphorylation induced by hyaluronan addition (not shown) or serum withdrawal was inhibited by high but not low concentration of okadaic acid (C-merlin in Fig. 1e; full-length merlin in Fig. 1f; merlin phosphorylation status was detected by a phospho-S518 antibody because full-length merlin resolves less well into two bands). Okadaic acid inhibits the protein serine/threonine phosphatase...

VprBP targets Merlin to the Roc1-Cul4A-DDB1 E3 ligase complex for degradation

“…Phosphorylation of Merlin at Ser518 was previously reported to play an important role in regulating Merlin tumor suppressor activity (Shaw et al, 2001;Alfthan et al, 2004;Jin et al, 2006a). Interestingly, the interaction between Merlin and VprBP is independent of Ser518 phosphorylation of Merlin (data not shown), suggesting that the Merlin-VprBP interaction might represent a new means for Merlin regulation in vivo.…”

“…Phosphorylation of a C-terminal serine (S518) by p21-activated kinase (PAK)-or cAMP-dependent protein kinase A weakens Merlin's self-association and is believed to inactivate its growth-suppressing activity. On the other hand, the phosphatase MYPT-1-PP1-d activates the tumor suppression activity of Merlin through S518 dephosphorylation, which subsequently inhibits the Ras/ERK (extracellular signal-regulated kinase) pathway (Shaw et al, 2001;Alfthan et al, 2004;Jin et al, 2006a). In addition, Merlin can also be regulated at the protein level by growth arrest stimuli.…”

Fibulin 1 is downregulated through promoter hypermethylation in gastric cancer

“…Its expression can suppress cell motility and inhibit the phosphorylation of extracellular signal-regulated kinase and myosin light chain (Twal et al, 2001). Moreover, FBLN1 can reduce the intracellular calcium level, which is important for the activation of multiple signal cascades such as the Ras/MAPK pathway (Twal et al, 2001;Jin et al, 2006Jin et al, , 2007. Interestingly, FBLN1 plays an important role in human papillomavirus (HPV) E6-mediated oncogenesis (Du et al, 2002).…”

Merlin: a tumour suppressor with functions at the cell cortex and in the nucleus

“…On this basis, it has been argued that Merlin mediates both contact inhibition and tumour suppression by directly modulating mitogenic signal transduction at or near the plasma membrane [21,22]. Analysis of various cell types indicated that Merlin can potentially affect a variety of mitogenic pathways, such as Rac-PAK signalling [7,[23][24][25], activation of mTORC1 independently of Akt [26,27], the EGFR-Ras-ERK path- [26,27], the EGFR-Ras-ERK path-,27], the EGFR-Ras-ERK pathway, the PI3K-Akt pathway and FAK-Src signalling [28][29][30][31]. In addition, genetic studies in Drosophila and mice showed that Merlin contributes to the activation of the Hippo tumoursuppressor pathway [18,32,33].…”