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SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

ABSTRACT: Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

SUBMITTER: Buchwald M

PROVIDER: S-EPMC3731393 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Buchwald M M Pietschmann K K Brand P P Günther A A Mahajan N P NP Heinzel T T Krämer O H OH

Oncogene 20121203 41

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protei ...[more]

PMID: 23208506

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Project description:Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of CRL4s or treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis. Abbreviations: ACTB, actin beta; ATG, autophagy-related; AMPK, AMP-activated protein kinase; AURKB/ARK2, aurora kinase B; BafA1, bafilomycin A1; CCNB1, cyclin B1; CDK1, cyclin dependent kinase 1; CHX, cycloheximide; CQ, chloroquine; CRL4s, CUL4-RING ubiquitin ligases; DDB1, damage specific DNA binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; STK11/LKB1,serine/threonine kinase 11; MTORC1/MTOR complex 1, mechanistic target of rapamycin kinase complex 1; NAE1, NEDD8 activating enzyme E1 subunit 1; NOC, nocodazole; RING, really interesting new gene; RBX1, ring-box 1; SA-GLB1/?-gal, senescence-associated galactosidase beta 1; TSC2, TSC complex subunit 2; TUBA, tubulin alpha; WIPI2, WD repeat domain, phosphoinositide interacting 2.

Dataset Information

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Publications

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

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