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

Oncogene volume 32, pages 4913–4920 (2013)Cite this article

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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.

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Acknowledgements

We thank C. Kosan, M. Korfei and S. Scheiding for their discussions and their excellent help. We are grateful to M. Kögl, N. Varin-Blank, Z. Ronai, R. Marschalek, H. Bursen, A. Baniahmad, O. Werz and O. Huber for providing material. Grant support: German Cancer Aid (FKZ102362); Wilhelm-Sander Foundation (No. 2010.078.1).

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  1. M Buchwald and K Pietschmann: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University, Jena, Germany

    M Buchwald, K Pietschmann, P Brand, T Heinzel & O H Krämer

  2. Department of Internal Medicine II, University of Giessen Lung Center, Giessen, Germany

    A Günther

  3. Drug Discovery Department, Moffitt Cancer Center, Tampa, FL, USA

    N P Mahajan

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Buchwald, M., Pietschmann, K., Brand, P. et al. SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation. Oncogene 32, 4913–4920 (2013). https://doi.org/10.1038/onc.2012.515

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