Ajuba Phosphorylation by CDK1 Promotes Cell Proliferation and Tumorigenesis

Chen, Xingcheng; Stauffer, Seth; Chen, Yuanhong; Dong, Jixin

doi:10.1074/jbc.m116.722751

Cited by 50 publications

(52 citation statements)

References 52 publications

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“…During these experiments, we found that MST2, but not MST1, was upshifted on a SDS-polyacrylamide gel during G2/M arrest (Fig. 1A, B) [16]. Lambda phosphatase treatment largely abolished the mobility upshift of MST2, suggesting that MST2 is phosphorylated during G2/M arrest (Fig.…”

Section: Resultsmentioning

confidence: 97%

“…Phos-tag ™ was obtained from Wako Pure Chemical Industries, Ltd. (304-93521) and used at 20 μM (with 100 μM MnCl 2 ) in 8% SDS-polyacrylamide gels as we previously described [16]. Western blotting, immunoprecipitation, and lambda phosphatase treatment assays were done as previously described [15,22].…”

Section: Methodsmentioning

confidence: 99%

“…We have recently shown that several upstream regulators (KIBRA and Ajuba) [14-16] and downstream effectors (YAP and TAZ) [17,18] of the Hippo pathway are phosphorylated during mitosis. During these previous studies, we found that the Hippo core kinase MST2 is also phosphorylated during antimitotic drug-induced G2/M phase arrest.…”

Section: Introductionmentioning

confidence: 99%

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MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity

Chen

Dong

2016

Cellular Signalling

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Mammalian sterile 20-like kinase 1/2 (MST1/2) are core tumor suppressors in the Hippo signaling pathway. MST1/2 have been shown to regulate mitotic progression. Here, we report a novel mechanism for phospho-regulation of MST2 in mitosis and its biological significance in cancer. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates MST2 in vitro and in vivo at serine 385 during antimitotic drug-induced G2/M phase arrest. This phosphorylation occurs transiently during unperturbed mitosis. Mitotic phosphorylation of MST2 does not affect its kinase activity or Hippo-YAP signaling. We further showed that mitotic phosphorylation-deficient mutant MST2-S385A possesses higher activity in suppressing cell proliferation and anchorage-independent growth in vitro and tumorigenesis in vivo. Together, our findings reveal a novel layer of regulation for MST2 in mitosis and its role in tumorigenesis.

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Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

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MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity

Chen

Dong

2016

Cellular Signalling

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“…Ajuba (Jub), a regulator of Aurora A kinase (36,37), was one of the strongest candidates in this cluster, with a 3-fold reduction in GSK-3-dependent phosphorylation at serine 129 (Fig. 3B), a site previously shown to be phosphorylated by other protein kinases to regulate entry into mitosis (37,38). Septin-9 (SEPT9), a member of the septin family of cytoskeletal proteins that regulates multiple functions, including cytokinesis and chromosome segregation (39,40), is phosphorylated at serine 85, also a GSK-3 consensus site, and this phosphorylation was reduced almost 2-fold in DKO ESCs.…”

Section: Gsk-3 Phosphoproteomementioning

confidence: 99%

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

Shinde¹,

Sidoli²,

Kulej³

et al. 2017

Journal of Biological Chemistry

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Edited by Eric R. FearonGlycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ϳ2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3␤ phosphorylated these proteins in vitro. RNASeq of WT and Gsk3 DKO ESCs identified ϳ190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ϳ47 proteins (1.4%) whose levels increased and ϳ78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. Glycogen synthase kinase-3 (GSK-3)2 is a ubiquitously expressed, highly conserved serine/threonine kinase that plays a central role in multiple signaling pathways, most prominently as an antagonist of insulin/AKT and Wnt/␤-catenin signaling pathways (1). Unlike most protein kinases, GSK-3 is constitutively active and in general is inhibited by upstream signaling. GSK-3 was first identified as a protein kinase that phosphorylates and inhibits glycogen synthase, the rate-limiting enzyme in glycogen synthesis (2) and was later shown to antagonize canonical Wnt signaling (1). However, GSK-3 is now known to regulate a broad range of cellular processes, including cytoskeletal organization, circadian rhythm, cell growth and survival, immune responses, and developmental processes (1).In metazoans, GSK-3 is encoded by two similar genes, Gsk3a and Gsk3b, with 98% amino acid sequence identity in the catalytic domains of mammalian GSK-3␣ and GSK-3␤. The two genes are partially redundant, and mice with complete loss of Gsk3a are viable due to compensation by Gsk3b. However, Gsk3b loss-of-function mutations in mice are embryonic or neonatal lethal (3,4), and the Gsk3a;Gsk3b DKO is lethal in early embryogenesis (5, 6). Furthermore, DKO mouse ESCs maintain expression of pluripotency markers and are unab...

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“…Phos-tag ™ was obtained from Wako Pure Chemical Industries, Ltd. (cat#: 304-93521) and used at 10–20 μM (with 100 μM MnCl 2 ) in 8% SDS-acrylamide gels as described [29]. Western blotting and lambda phosphatase treatment assays were done as previously described [25].…”

Section: Methodsmentioning

confidence: 99%

CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity

Stauffer

Zeng

Zhou

et al. 2017

Cellular Signalling

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PDZ-binding kinase (PBK) plays a major role in proliferation and in safeguarding mitotic fidelity in cancer cells. Frequently upregulated in many cancers, PBK drives tumorigenesis and metastasis. PBK has been shown to be phosphorylated in mitosis by cyclin-dependent kinase 1 (CDK1)/cyclin B, however, no studies have been done examining PBK mitotic phosphorylation in oncogenesis. Additionally to the previously identified Threonine-9 phosphorylation, we found that Threonine-24, Serine-32, and Serine-59 of PBK are also phosphorylated. PBK is phosphorylated in vitro and in cells by CDK1 during antimitotic drug-induced mitotic arrest and in normal mitosis. We demonstrated that mitotic phosphorylation of Threonine-9 is involved in cytokinesis. The non-phosphorylatable mutant PBK-T9A augments tumorigenesis to a greater extent than wild type PBK in breast cancer cells, suggesting that PBK mitotic phosphorylation inhibits its tumor promoting activity. The PBK-T9A mutant also transforms and increases the proliferation of immortalized breast epithelial cells. Collectively, this study reveals that CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity.

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Ajuba Phosphorylation by CDK1 Promotes Cell Proliferation and Tumorigenesis

Cited by 50 publications

References 52 publications

MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity

MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity

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