br Introduction ACK or Activated Cdc Associated Kinase locat

Introduction ACK1, or Activated Cdc42-Associated Kinase, located on chromosome 3q, is a ubiquitously expressed non-receptor tyrosine kinase cloned from a human Bryostatin 1 mg cDNA library (Manser et al., 1993). It was first identified to bind to activated Cdc42, a small Ras GTPase via its CRIB domain (Manser et al., 1993). This interaction activates ACK1 via autophosphorylation which subsequently activates Dbl, a Rho guanine exchange factor (Kato et al., 2000) as well as p130cas (Modzelewska et al., 2006). Dbl in turn activates the Rho family of GTPases, resulting in cytoskeletal rearrangements. In the last few years, reports have shown that Rho family proteins play an important role in tumor progression (Rosel et al., 2008; Sequeira et al., 2008; Tang et al., 2008; Vega and Ridley, 2008). In addition to activated Cdc42, other ACK1 interacting partners include clathrin, Grb2, EGFR, ubiquitin and Nedd4-2 E3 ligase (Chan et al., 2009; Kato-Stankiewicz et al., 2001; Shen et al., 2007; Teo et al., 2001). Its association with clathrin has been proposed to be involved in receptor-mediated endocytosis (Shen et al., 2007). ACK1 is shown to bind EGFR in a Grb2-dependent manner and aids EGFR downregulation with its functional ubiquitin association domain (UBA) (Grovdal et al., 2008; Kato-Stankiewicz et al., 2001; Shen et al., 2007). Recently, ACK1 has been implicated in cancer progression. Gene amplification of ACK1 was reported in several cancer types such as lung-, prostate- and ovarian tumors (van der Horst et al., 2005), while cDNA microarrays demonstrated an upregulation of ACK1 expression in gastric carcinoma (Wang et al., 2006). ACK1 is also associated with androgen-independent prostate cancer (Mahajan et al., 2007) and aids in tumor metastasis through its positive activation of p130cas which, localized to the focal adhesion sites, responses to integrin signaling and is involved in spreading of melanoma cells (Eisenmann et al., 1999). ACK1 also exerts an oncogenic function through tumor suppressor, Wwox phosphorylation, targeting it for polyubiquitination and subsequent degradation (Mahajan et al., 2005). In LnCaP and 4T1 xenograft models, ACK1-overexpressing cells give rise to aggressive metastatic tumors (Mahajan et al., 2007; van der Horst et al., 2005). Silencing of the ACK1 gene in Ras-transformed NIH3T3 cells on the other hand increases apoptosis (Nur et al., 2005). Previously, we reported the detection of several novel somatic- and germ line mutations in the coding sequence of ACK1 of different cancer cell lines and control tissues (Ruhe et al., 2007). In this paper, we characterized two somatic mutations, A634 T and S985 N in KatoIII and A498 cells, respectively. A634 T is located in the newly established E3 ligase binding domain (Chan et al., 2009) while S985 N is found in the UBA domain. We rationalized that the location of the mutations may confer extended protein stability to the kinase, thereby unleashing an inherent oncogenic activity. Our data demonstrate that the S985 N mutation in ACK1 confers enhanced proliferative and migratory properties as well as anchorage-independent growth. Mechanistic studies revealed that the ACK1 S985 N mutation has lost ubiquitin interaction, which in turn, enhances kinase stability, delays ubiquitination and reduces downregulation of EGFR. Interestingly, silencing of ACK1 S985 N sensitizes renal carcinoma cells to gefitinib. These findings not only provide us with new insights into mechanisms that underlie ACK1-mediated oncogenesis but also highlight the possibility of combination therapy by targeting ACK1 in cancer cells that are previously resistant to EGFR inhibitors.
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Results
Discussion The broad importance of PTKs in tumorigenesis has been demonstrated over many years. Oncogenic mutations have been identified in clinical samples as well as cancer cell lines [reviewed in (Zhang et al., 2009)]. In our work, we addressed the question if an oncogenic role is conferred on ACK1 by a change in its stability. We characterized two missense mutations which resulted in a single amino acid change at the E3 ligase binding domain and UBA in gastric and renal cancer cells, respectively. In this study, the effect of the somatic mutation was analyzed using MCF7 stable clone overexpression and gene silencing in A498 cells. We demonstrated that the ACK1 S985 N mutation enhances cell proliferation, migration, anchorage-independent growth as well as mitogenic signaling, compared to the wild type kinase. Interestingly, silencing of ACK1 (S985 N) in A498 cells resulted in an upregulation of epithelial markers suggestive of EMT reversion. Our results also demonstrated that ACK1 with a mutation at residue 985 loses its ability to bind ubiquitin, which stabilized the kinase and impaired its role in EGFR downregulation and degradation through receptor ubiquitination and proteasomal degradation.