Introduction Mammalian cells express the seven STAT family m

Introduction Mammalian LDN 57444 synthesis express the seven STAT family members STAT1, −2, −3, −4, −5A, −5B, and −6 [1], [2]. All STATs exert physiologically important roles as homo- and heterodimers [2], [3], [4]. Cytokines and growth factors activate STATs through the activation of kinases that phosphorylate serine and tyrosine residues in the C-terminal domains of STATs [2], [5]. Additional posttranslational modifications critically control the activity of STATs [3], [6], [7], [8], [9]. Tyrosine phosphorylation is the best-characterized and most activating posttranslational modification of STATs. Upon binding of a cognate ligand to its receptor, receptor-associated JAKs (JAK1–3 and TYK2) catalyse the phosphorylation of STATs. This leads to an avid interaction between the phosphorylated tyrosine and the Src homology-2 (SH2) domains of two STAT molecules and the nuclear import of such dimers [1], [2]. These attach to specific chromatin regions, where they recruit the transcription machinery to activate gene expression [1], [2], [6]. The N-terminal domains of STATs confer a weaker, but detectable interaction of STATs in vivo[2], [3], [4]. Recent evidence shows that ACK1 is a further kinase that can induce the tyrosine phosphorylation of STAT1 and subsequent STAT1-dependent gene expression in liver cells [10]. Like JAKs, ACK1 is a mammalian non-receptor tyrosine kinase that contributes to important physiological processes and to severe human diseases including cancer [11]. Cytokines like epidermal growth factor and platelet-derived growth factor as well as integrin β1 activation are among the stimuli that promote the phosphorylation-dependent activation of ACK1 and subsequent downstream signalling [12]. Various pathways control the stability of ACK1. These include lysosomal and proteasomal degradation pathways that involve the E3 ubiquitin ligases neural-precursor-cell-expressed, developmentally-downregulated and the seven-in-absentia homologues SIAH1/SIAH2, respectively [11], [12], [13], [14]. HSP90β also regulates the activity of ACK1 and its pro-tumourigenic functions [15]. ACK1 phosphorylates and activates STAT1 in liver-derived cells [10], but it has not been resolved which regulators of ACK1 modulate this effect. Furthermore, ACK1 might also phosphorylate other STATs. Here we demonstrate that catalytically active ACK1 induces the tyrosine phosphorylation of STAT1 and STAT3. We additionally show that active HSP90 is required for the ACK1-dependent phosphorylation of STAT1/STAT3 in cultured human cells. Furthermore, our data suggest that ACK1 phosphorylates STAT3 in primary lung cancer samples.
Material and methods
Results
Discussion Our work demonstrates that catalytically active ACK1 can promote the activation of STAT1 and STAT3. A degradation of ACK1 by SIAH2 as well as the pharmacological inhibition of HSP90 with AT13387 suppress the phosphorylation of STAT1 and STAT3. We summarize these new findings in Fig. 5E. We collected the data in a permanent human cell line and primary ADC samples. As methods, we used reporter gene assays, immunoblot, biochemical fractionation assays, IHC, and IP techniques. It was previously reported that ACK1 could interact with overexpressed STAT1 in the human hepatoma cell line Huh7. The authors detected an ACK1-dependent phosphorylation of STAT1 when they enriched STAT1 by IP [10]. We show that an ACK1-dependent tyrosine phosphorylation of endogenous STAT1 can be detected in whole cell extracts of HEK293T cells. In addition, we demonstrate that ACK1 promotes the phosphorylation of endogenous STAT3 and the nuclear accumulation of p-STAT1 and p-STAT3. Congruent with these data, an inactivation of ACK1 with the tyrosine kinase inhibitor Dasatinib attenuates p-STAT3 [29]. Further studies will address whether ACK1 also phosphorylates STAT5, which is frequently dysregulated in ADC [30]. The above data are consistent with the finding that SIAH2 targets ACK1 for proteasomal degradation [13]. We reported that SIAH2 was overexpressed in primary squamous cell carcinoma of the lung [31] and others verified this finding in an independent cohort [32]. This overexpression of SIAH2 is associated with a reduced expression of TYK2, attenuated phosphorylation of STAT3, and a lower expression of its target matrix metalloproteinase-1 [31]. Therefore, SIAH2 may target ACK1 and consequently the phosphorylation and activation of STAT1 and STAT3 in lung cancer. The relevance of this finding could be identified in vivo and may be more complex than anticipated. While STAT3 can be a poor prognostic factor in lung cancer patients [33], STAT3 can equally act as tumour suppressor in lung and prostate cancer cells [34], [35]. Furthermore, SIAH2 and ACK1 may control prostate cancer cell growth [12], [14] through their antagonistic regulation of STAT-dependent gene expression.