title: 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.
authors.affiliation: Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University, Jena, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Buchwald
authors.forename: K
authors.initials: K
authors.lastname: Pietschmann
authors.forename: P
authors.initials: P
authors.lastname: Brand
authors.forename: A
authors.initials: A
authors.lastname: Günther
authors.forename: N P
authors.initials: NP
authors.lastname: Mahajan
authors.forename: T
authors.initials: T
authors.lastname: Heinzel
authors.forename: O H
authors.initials: OH
authors.lastname: Krämer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D017124:Conserved Sequence; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000098563:Seven in Absentia Proteins
chemical_list: D009687:Nuclear Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; D046988:Proteasome Endopeptidase Complex