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Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis. [ABSTRACT FROM AUTHOR]

Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,KATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr545. Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts withMST4and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr545 by mass spectrometric analyses. Importantly, phosphorylation of Thr545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylationdeficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr545 enables ACAP4 to interact with ezrin. Given the location of Thr545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells. [ABSTRACT FROM AUTHOR]

Telomerase activation plays a critical role in human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. The human telomerase reverse transcriptase (hTERT) promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. Deregulation of the Wnt/β- catenin signaling pathway is reported to be associated with human carcinogenesis. However, little is known about whether the Wnt/β-catenin pathway is involved in activating hTERT transcription and inducing telomerase activity (TA). In this study, we report that hTERT is a novel target of the Wnt/β- catenin pathway. Transient activation of the Wnt/β-catenin pathway either by transfection of a constitutively active form of β-catenin or by LiCl or Wnt-3a conditioned medium treatment induced hTERT mRNA expression and elevated TA in different cell lines. Furthermore, we found that silencing endogenous β-catenin expression by β-catenin gene-specific shRNA effectively decreased hTERT expression, suppressed TA, and accelerated telomere shortening. Of the four members of the lymphoid- enhancing factor (LEF)/T-cell factor (TCF) family, only TCF4 showed more effective stimulation on the hTERT promoter. Ectopic expression of a dominant negative form of TCF4 inhibited hTERT expression in cancer cells. Through promoter mapping, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we found that hTERT is a direct target of β-catenin·TCF4-mediated transcription and that the TCF4 binding site at the hTERT promoter is critical for β-catenin·TCF4-dependent expression regulation. Given the pivotal role of telomerase in carcinogenesis, these results may offer insight into the regulation of telomerase in human cancer. [ABSTRACT FROM AUTHOR]