These data demonstrate that NPM-ALK kinase activity is required for the serine-9 phosphorylation of GSK3. Interrogation of the phosphoproteomic data identified several serine kinases that are known to phosphorylate GSK3 Aceneuramic acid hydrate (data not shown). in 293T cells led to an increase of pS9-GSK3 (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3 levels. Phosphorylation of pS9-GSK3 by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3 substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3 inhibition. Furthermore, stable knockdown of GSK3 conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS9-GSK3 and CDC25A were selectively expressed in neoplastic cells of ALK + ALCL tissue biopsies, and showed a significant correlation (P< 0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS9-GSK3 and CDC25A expression (P< 0.2). Our results demonstrate Rabbit Polyclonal to ECM1 that NPM-ALK regulates the phosphorylation of S9-GSK3 by PI3K/AKT. The subsequent inhibition of GSK3 activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3 as a mediator of NPM-ALK oncogenesis. Keywords:NPM-ALK, GSK3, ALCL, oncogenesis == Introduction == Anaplastic large cell lymphoma (ALCL) is an aggressive form of non-Hodgkin lymphoma often characterized by the chromosomal translocationt(2;5)(p23;q35). This translocation results in the expression of the nucleo-phosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein (Morriset al., 1994). Although the expression ofALKis usually confined to neonatal brain tissue (Iwaharaet al., 1997), the translocation places Aceneuramic acid hydrate the gene under the regulation of theNPMpromoter, which is abundantly and ubiquitously expressed. Furthermore, the dimerization of the fusion protein mediated by the oligomerization domain of NPM (Bischofet al., 1997) leads to its autophosphorylation and constitutive activation (Amin and Lai, 2007;Palmeret al., 2009). NPM-ALK activates diverse growth factor signaling pathways including the JAK/STAT (Zamoet al., 2002), the PI3K/AKT (Slupianeket al., 2001) and the PLC pathways (Baiet al., 1998). However, the cellular mechanisms utilized by NPM-ALK to induce oncogenesis have not been completely characterized. To identify novel signaling proteins regulated by NPM-ALK, we carried out phosphoproteomic studies oft(2;5) + cell lines using the approach outlined previously (Rushet al., 2005). Our studies provide evidence that GSK3 is a novel NPM-ALK-regulated protein. GSK3 is a serine/threonine kinase that is constitutively active in resting cells (Cohen and Frame, 2001;Jope and Johnson, 2004). Upon growth factor signaling, GSK3 is phosphorylated at serine-9, which inhibits its ability to target key substrates for proteasomal degradation. The accumulation of substrates of GSK3 such as CDC25A and Mcl-1 that regulate the cell cycle and apoptosis contributes to the enhanced proliferative capacity of tumor cells (Jinnoet al., 1994;Diehlet al., 1997;Zhaoet al., 2007;Kanget al., 2008). Here, we show that serine-9 phosphorylation of GSK3 is dependent on NPM-ALK kinase activity and the PI3K/AKT pathway. We provide evidence that NPM-ALK results in stabilization of Mcl-1 and CDC25A through GSK3 inhibition, which in turn enhances cell viability, proliferation and oncogenic potential. Furthermore, tissue biopsies of ALK + ALCLs express pS9-glycogen synthase kinase 3 beta (GSK3) as well as CDC25A, providing additional support for its functional significance in ALK + ALCLs. == Results and discussion == Given the oncogenic role of constitutively active NPM-ALK tyrosine kinase in ALK + ALCL, Aceneuramic acid hydrate we hypothesized that NPM-ALK-expressing cell lines would exhibit a phosphoproteomic signature that would reflect the signaling cascade regulated by the oncogene. To gain insight into these signaling pathways and to identify novel regulators of NPM-ALK-induced oncogenesis, we used a phosphoproteomic approach (Rushet al., 2005) to evaluate the tyrosine phosphorylated protein in the NPM-ALK-expressing SUPM2 cell series. SUPM2 cell lysates had been put through phosphopeptide enrichment using phosphotyrosine-specific antibodies, accompanied by tandem mass spectrometry. NPM-ALK-derived phosphotyrosine peptides furthermore to numerous various other phosphorylated proteins had been identified (data not really proven). Among this list was an eleven amino-acid tryptic peptide matching to GSK3 proteins phosphorylated at Y216 (Statistics 1a and b). Of be aware, this phosphotyrosine peptide continues to be identified in an identical study adding to the self-confidence of this applicant proteins (Boccalatteet al., 2009). Based on the known function of GSK3 in mobile signaling (Jope and Johnson, 2004), we hypothesized that GSK3 might mediate the oncogenic properties of NPM-ALK. As GSK3 activity may be governed by growth aspect signaling through serine9phosphorylation, we hypothesized that NPM-ALK regulates the serine phosphorylation of GSK3 and inhibits its activity. == Amount 1. ==.