Interestingly, the AMPK1 T19A, and combined T19A and S40A (2A) mutations blocked all 32P incorporation at both T19 and S40, possibly indicating that lack of T19 phosphorylation precludes S40 phosphorylation, but not vice versa (Fig
Interestingly, the AMPK1 T19A, and combined T19A and S40A (2A) mutations blocked all 32P incorporation at both T19 and S40, possibly indicating that lack of T19 phosphorylation precludes S40 phosphorylation, but not vice versa (Fig.?2F). in promoting proper chromosomal alignment, as loss of AMPK activity leads to misaligned chromosomes and concomitant metaphase delay. Importantly, AMPK expression and activity was found to be critical for paclitaxel chemosensitivity in breast cancer cells and positively correlated with relapse-free survival in systemically treated breast cancer patients. cells have mitotic defects (Lee et al., 2007). AMPK has also been shown to be activated during mitosis, with increased p-T172 phosphorylation seen during mitosis (Vazquez-Martin et al., 2009, 2012; Thaiparambil et al., 2012; Mao et al., 2013; Lee et al., 2015; Domnech et al., 2015). Likewise, a screen Rabbit Polyclonal to HDAC5 (phospho-Ser259) of AMPK substrates revealed multiple downstream mitotic proteins as targets of its kinase activity (Banko et al., 2011). A chemical genetic screen of downstream AMPK substrates in human cells identified several that were involved in mitosis, including protein phosphatase 1 regulatory subunit 12A and 12C (PPP1R12A and PPP1R12C), cell division cycle protein 27 (CDC27), and p21-activated protein kinase (PAK2) (Banko et al., 2011). AMPK phosphorylation of PPP1R12C blocks its inhibition of myosin regulatory light chain proteins, (MRLCs), which are regulators of cytokinesis (Ito et al., 2004), CDC27 is a Mirabegron member of the APC connecting AMPK to the spindle checkpoint during metaphase (Peters, 2006), and AMPK activation of PAK2 leads to phosphorylation of MRLCs and mitotic progression (Tuazon and Traugh, 1984). MRLCs have also been shown to be phosphorylated directly by AMPK at their regulatory site and and mammals (Mirouse et al., 2007). AMPK has been connected to mitosis in other studies as well. AMPK-null embryos display severe abnormalities in cytoskeletal apicalCbasal polarity, as well as defective mitotic divisions that lead to polyploidy (Lee et al., 2007). Loss of AMPK activity, through either inhibition of AMPK in cancer cells (Sanli et al., 2010) or with full AMPK knockout (KO) in mouse embryonic fibroblasts (MEFs) (Sanli et al., 2012), is enough to weaken the cell cycle arrest at G2/M caused by ionizing radiation. Interestingly, due to the important role microtubules play in mitotic cell division, inhibition of AMPK has been shown to impair microtubule stabilization through Mirabegron Mirabegron loss of phosphoregulation of the microtubule plus-end protein CLIP-170 (also known as CLIP1) (Nakano et al., 2010). There is evidence that CLIP-170 itself mediates paclitaxel sensitivity in breast cancer cells through its ability to strengthen microtubule assembly promoted by paclitaxel (Sun et al., 2012). AMPK is also active in the mitotic regulation of neural stem cells. Abolishing normal AMPK activity in the developing mouse brain leads to flawed mitosis in neural progenitor cells and abnormal brain development (Dasgupta and Milbrandt, 2009). Recently, it has been discovered that AMPK and its ortholog Snf1 in are required for proper metaphase spindle alignment (Thaiparambil et al., 2012; Tripodi et al., 2018). Together, these studies point to a role for AMPK outside of its canonical signaling network, acting as a master regulator not only of cellular metabolism, but also cell cycle progression. Despite AMPK’s connection to mitosis, how AMPK is regulated during mitotic progression remains unclear. In this report, we identify a novel layer of regulation involving CDK1-mediated phosphorylation for AMPK. RESULTS AMPK is phosphorylated during anti-tubulin drug-induced mitotic arrest To examine the phosphorylation status of the AMPK subunits, we used PhosTag gel electrophoresis which selectively separates phosphorylated from unphosphorylated proteins through specific binding of phosphate ions (see Zhang et al., 2015, Stauffer et al., 2017). The mobility shifts of AMPK1, AMPK2 and AMPK1 (also known as Mirabegron PRKAA1, PRKAA2 and PRKAB1, respectively) were seen to be increased during mitotic arrest induced by anti-mitotic drugs (Fig.?1A), suggesting that AMPK is phosphorylated during mitotic arrest. The mobility of AMPK2, AMPK1, AMPK2 and AMPK3 (also known as PRKAB2, PRKAG1, PRKAG2 and PRKAG3, respectively) were not altered under these conditions (Fig.?1A). We found that the phosphorylation levels of AMPK1 and AMPK2 at the main T172 activation site and AMPK1 at S108 and S182 were not changed under these conditions. This suggests that the mobility shift of AMPK was not likely due to phosphorylation at T172 or S108/S182 respectively and indicates the possibility of novel post-translational modification sites (Fig.?1B). Treatment of arrested cells with -phosphatase completely reversed the mobility shift Mirabegron of AMPK and AMPK1 (Fig.?1C), indicating that the mobility shifts of AMPK subunits during mitosis were due to phosphorylation events. In order to determine which upstream kinases could be phosphorylating AMPK, we took cells that were cultured overnight with taxol and then treated for 2 h with various kinase inhibitors. Interestingly, only the CDK1 inhibitors.