Merlin Mediates PICT-1-induced Growth Inhibition Of Glioblastoma Cells In The Nucleus

Merlin, encoded by the NF2gene, belongs to the ERM protein family. Previousresearches showed that Merlin is mainly distributed at the cellular cortex and functionsas a tumor suppressor through inhibiting mitogenic signalling at the plasmamembrane-cytoskeleton interface. However, the cell cycle-dependentnucleocytoplasmic shuttling of Merlin has been observed in a recent study, suggestingthat Merlin may also exerts its tumor suppressor function in the nucleus by interactingwith specific nuclear protein partners. Here, we identified PICT-1as a novelMerlin-binding partner by yeast two-hybrid screen. Though some evidences suggestedthat PICT-1is a candidate tumor suppressor, the exact mechanisms by which PICT-1suppresses cell growth are not fully understand. Previous studies showed that PICT-1suppressed cell growth and promoted apoptosis in a PTEN-dependedent maner bybinding to PTEN and regulate its phosphorylation and stability. In the present research,confocal fluorescent microscopy and nuclear co-immunoprecipitation showed PICT-1was a nucleolar-localized protein and Ser518-dephosphorylated Merlin (the growthinhibitory form of Merlin) preferentially co-localized and interacted with PICT-1in thenucleolus following ectopic over-expression of PICT-1. PICT-1over-expression both inPTEN-positive HeLa cells and in PTEN-deficient U251cells could efficiently represscyclin D1expression, arrest cell cycle at G0/G1and promote cell apoptosis. The1-356fragment of PICT-1, a carboxyl-terminus deleted mutant that has lost theMerlin-binding ability, had a markedly reduced inhibitory effect. Merlin knockdown byspecific siRNA also attenuated the inhibitory effects of PICT-1over-expression,whereas PICT-1knockdown did not significantly change the inhibitory effects ofMerlin over-expression. Thus PICT-1function as a tumor suppressor was showed to beat least partially dependent on Merlin expression, though restoration of PTEN functionfurther promoted the apoptosis of U251cell induced by PICT-1over-expression.Collectively, we propose that in exception to its tumor suppressor function atplasma membrane level, Merlin also mediates PICT-1-induced growth inhibition bytranslocating to the nucleolus and binding to PICT-1.