Regulation of ACK1 Activity
ACK1 is a multidomain nonreceptor tyrosine kinase expressed in a variety of tissues in mammals. In addition to the N-terminally located kinase catalytic domain, ACK1 contains a membrane-targeting sterile alpha motif domain, a Src homology 3 (SH3) domain, a Cdc42/Rac interactive binding domain, a clathrin-binding motif, a Mig6 homology domain, a proline-rich domain (which overlaps with the clathrin-binding motif and the Mig6 homology domain), and a ubiquitin-association domain (Fig. 1). These domains are responsible for recognition and interaction of diverse binding partners (Mahajan and Mahajan 2010).
Originally, ACK1 was identified as a specific target of Cdc42: The active GTP-bound, but not inactive GDP-bound, Cdc42 interacts with the Cdc42/Rac interactive binding domain of ACK1. Thus, Cdc42 is expected to have a crucial role in the regulation of ACK1. Although coexpression of Cdc42 in cells results in tyrosine phosphorylation of ACK1, the binding of Cdc42·GTP to purified ACK1 was found to be insufficient for stimulating its tyrosine kinase activity in vitro (Yang et al. 1999; Yokoyama and Miller 2003). Given that many signaling molecules interact with ACK1, Cdc42 may not be a unique upstream regulator, but rather may collaborate with other regulatory proteins to modulate the activity of ACK1 in the cell.
Many protein kinases are catalytically activated through conformational change of the activation loop triggered by its phosphorylation. Therefore, ACK1 kinase activity may also be modulated by phosphorylation of the activation loop. One tyrosine (Y284, all residue numbers hereafter are based on the mouse ACK1 sequence [NCBI accession: NP_058068]), which exists within the activation loop of ACK1, was identified as a primary autophosphorylation site (Yokoyama and Miller 2003). Comparison of tertiary structures of phosphorylated and unphosphorylated activation loops of ACK1, unexpectedly, revealed that ACK1 adopts an activated conformation independent of phosphorylation of Y284 (Lougheed et al. 2004). Therefore, the activation loop of ACK1 is not autoinhibitory, although it may have another role in the regulation of catalysis.
Recently, the intramolecular association between the N-terminal kinase catalytic domain and the C-terminal Mig6 homology domain, which may suppress the kinase activity, has been revealed (Prieto-Echagüe et al. 2010). In fact, amino acid changes in kinase catalytic (E346K) and Mig6 homology (F836A) domains disrupt this interaction and markedly increase the kinase activity (Prieto-Echagüe et al. 2010). In fact, the former mutation has been characterized as a constitutively activated mutant in cancer cells (see below). Furthermore, Grb2-mediated activation of ACK1 by releasing the autoinhibition in response to epidermal growth factor (EGF) stimulation was proposed (Lin et al. 2012). Arguing against this autoinhibition model, Src-dependent phosphorylation of Y284 and subsequent activation of ACK1 was also reported (Chan et al. 2011).
Although the underlying mechanism remains totally unknown, the tyrosine phosphorylation level of ACK1 significantly increased in response to temperature shift-down to 25°C and hyperosmotic shock (Satoh et al. 1996).
ACK1 in Cell-Surface Receptor Signaling
Melanoma chondroitin sulfate proteoglycan is a cell-surface antigen that stimulates integrin-α4β1-mediated adhesion and spreading of melanoma cells. Clustering of this antigen induces the activation of Cdc42 and ACK1, leading to tyrosine phosphorylation of p130Cas, a key molecule for the induction of tumor cell motility and invasion (Eisenmann et al. 1999). Therefore, ACK1 may play an important role in the regulation of melanoma chondroitin sulfate proteoglycan-dependent melanoma cell migration and invasion. ACK1 is also activated upon cell attachment to fibronectin in a Cdc42-dependent manner, suggesting a role in outside-in signaling of integrins (Yang et al. 1999; Galisteo et al. 2006). Stimulation of the M3 muscarinic acetylcholine receptor also triggers the activation of ACK1 (Linseman et al. 2001). Neither the increase in intracellular Ca2+ nor the activation of protein kinase C is required for this ACK1 activation. Instead, Cdc42 and the tyrosine kinase Fyn may be involved. ACK1 is also involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor recruitment to lipid rafts and TRAIL-induced cell death (Linderoth et al. 2013). Interestingly, ACK1 kinase activity is not required for this function.
ACK1 in Endocytosis and Protein Degradation
ACK1 also participates in the regulation of ligand-induced endocytosis and degradation of the receptor (Fig. 2). ACK1 possesses a conserved clathrin-binding motif, which in fact interacts with the N-terminal head region of the clathrin heavy chain (Teo et al. 2001; Yang et al. 2001). Activated Cdc42 negatively regulates this interaction. When overexpressed, ACK1 competes with AP-2 for the binding to clathrin, and thus suppresses clathrin-mediated endocytosis of the transferrin receptor. Furthermore, ACK1 directly binds to sorting nexin 9 (SH3PX1) through the interaction between the proline-rich domain of ACK1 and the SH3 domain of sorting nexin 9, thereby facilitating endocytosis of the EGF receptor. EGF-induced degradation of the EGF receptor is mediated by ubiquitination. ACK1 binds to the ubiquitinated EGF receptor through its ubiquitin-association domain, thereby regulating ligand-induced EGF receptor degradation (Shen et al. 2007). In addition, ACK1 is ubiquitinated by E3 ubiquitin ligases Nedd4-1 and Nedd4-2 and subjected to degradation via the lysosomal pathway along with the EGF receptor in response to EGF stimulation. ACK1 is also ubiquitinylated by SIAH ubiquitin ligases, and degradation of ACK1 in the proteasomal pathway is facilitated in estrogen-stimulated breast cancer cells (Buchwald et al. 2013). Cortactin is a newly identified substrate of ACK1 and plays a role during ligand-mediated EGF receptor internalization (Kelley and Weed 2012).
In presynaptic neurons, ACK1 is involved in the regulation of plasma membrane expression of the dopamine transporter, which facilitates high-affinity presynaptic dopamine reuptake, and thereby temporally and spatially modulates dopamine neurotransmission (Wu et al. 2015). Cdc42-activated ACK1 specifically inhibits dopamine transporter endocytosis, and thereby stabilizes the dopamine transporter at the plasma membrane. Protein kinase C-dependent inactivation of ACK1 in turn facilitates rapid clathrin- and dynamin-dependent dopamine transporter internalization and intracellular sequestration (Wu et al. 2015).
ACK1 as a Link Between Small GTPases
Another GEF whose activity is regulated by ACK1 is Ras-GRF1 (CDC25Mm (mammalian homologue of cell division cycle 25)) (Kiyono et al. 2000). Ras-GRF1 targets both Ras and Rac1 through its CDC25 homology and Dbl homology domains, respectively. The latent GEF activity toward Rac1 is induced by G protein βγ subunit- and Src-dependent tyrosine phosphorylation of Ras-GRF1 although its Ras GEF activity remains unchanged. In contrast, coexpression of activated ACK1 causes tyrosine phosphorylation of Ras-GRF1 and promotes its GEF activity toward Ras. Through this mechanism, ACK1 links Cdc42 to Ras. Tyrosine phosphorylation of Ras-GRF1 by ACK1, on the other hand, does not induce Rac1 GEF activity. Taken together, different tyrosine kinases including ACK1 phosphorylate different residues of Ras-GRF1, leading to selective induction of GEF activity toward Ras or Rac1.
ACK1 in Cancer
The involvement of ACK1 in prostate cancer progression has been well described. Expression and tyrosine phosphorylation levels of ACK1 are elevated in clinical specimens of androgen-independent prostate cancer (Mahajan and Mahajan 2010; van der Horst et al. 2005). A constitutively activated ACK1 mutant, when expressed in a human prostatic adenocarcinoma cells, remarkably promotes anchorage-independent growth and tumor formation in nude mice (Mahajan and Mahajan 2010). Transgenic mice expressing this constitutively activated ACK1 in the prostate indeed develop prostatic intraepithelial neoplasia (Mahajan and Mahajan 2010).
One mechanism for ACK1-dependent promotion of prostate tumorigenesis is negative regulation of the proapoptotic tumor suppressor WW domain-containing oxidoreductase (Mahajan and Mahajan 2010). Activated ACK1 associates with and tyrosine phosphorylates WW domain-containing oxidoreductase, leading to its polyubiquitination followed by degradation.
Another mechanism is tyrosine phosphorylation of the androgen receptor by ACK1 (Mahajan et al. 2007). This phosphorylation of the androgen receptor may be important in androgen-independent progression of prostate cancer. Activated ACK1 phosphorylates two tyrosine residues in the transactivation domain of the androgen receptor, leading to the recruitment of the androgen receptor to the androgen-responsive enhancer and subsequent androgen-inducible gene expression in the absence of androgen. Thereby, activated ACK1 promotes androgen-independent growth of prostate xenograft tumors (Mahajan et al. 2007). On the other hand, ACK1 is required for tyrosine phosphorylation and activation of the androgen receptor by ligand-activated HER2 as evidenced by the effect of knockdown and the treatment with a specific inhibitor, dasatinib (Mahajan et al. 2007; Liu et al. 2010). Therefore, ACK1 activation downstream of cell-surface receptor tyrosine kinases such as HER2 may be a crucial event in prostate cancer cells. Another ACK1-specific inhibitor, termed AIM-100, also suppresses phosphorylation and transcriptional activation of the androgen receptor (Mahajan and Mahajan 2010). In breast tumors, HER2-mediated activation of ACK1 is reported to be responsible for estrogen-independent estrogen receptor-regulated gene transcription (Mahajan et al. 2014). Activated ACK1 phosphorylates the histone demethylase KDM3A, thereby promoting transcription of an estrogen-regulated gene even in an estrogen-deficient environment.
The activation of the prosurvival protein kinase Akt has been implicated in a variety of human cancers. It has been well documented that Akt activation occurs through specific phosphorylation of serine and threonine residues downstream of phosphatidylinositol 3-kinase. Recently, a novel signaling mechanism whereby ACK1 directly regulates Akt by phosphorylating a tyrosine residue located in the kinase domain (Mahajan and Mahajan 2010). Upon tyrosine phosphorylation, Akt is translocated to the plasma membrane and then activated. This mechanism, in fact, has become relevant to human cancers. The expression level of Akt that is phosphorylated by ACK1 is significantly increased in breast cancers and is correlated with the severity of disease progression.
Four somatic missense mutations were identified in ACK1 in various cancers: Mutations in the N-terminal sterile alpha motif domain (R34L and R99Q) were identified in lung adenocacinoma and ovarian mucinous carcinoma, respectively, a mutation in the kinase catalytic domain (E346K) was found in ovarian endometrioid carcinoma, and a mutation in the SH3 domain (M409I) was identified in gastric adenocacinoma. All these mutations increase ACK1 kinase activity, suggesting that somatic mutations may represent a mechanism for oncogenic activation of ACK1 (Prieto-Echagüe et al. 2010). Several somatic and germ-line mutations were detected also in tumor cell lines. Among them, a mutation in the ubiquitin-association domain (S1002N) renders ACK1 unable to bind ubiquitin and maintains the epidermal growth factor receptor level after stimulation. Thereby, this ACK1 mutant may contribute to prolonged mitogenic signaling in cancer cells.
ACK1 is a nonreceptor tyrosine kinase originally identified as a target of the small GTPase Cdc42. ACK1 interacts with diverse signal transducing proteins through multiple domains. It is proposed that the kinase activity of ACK1 is suppressed by the intramolecular interaction between the catalytic domain and the C-terminal Mig6 homology domain. Upstream signals are believed to induce conformational change of inactive ACK1, leading to its activation. ACK1 functions downstream of a variety of receptor tyrosine kinases such as the EGF receptor. In addition, other types of cell-surface receptors, including melanoma chondroitin sulfate proteoglycan and integrins, employ ACK1 as a transducer of signals that regulate cell motility. ACK1 is also responsible for ligand-induced endocytosis of cell-surface receptors through the binding to the clathrin heavy chain and sorting nexin 9. ACK1 is ubiquitinated by E3 ubiquitin ligases and subjected to degradation. ACK1 acts as a link between small GTPases by phosphorylating GEFs, such as Dbl and Ras-GRF1. Gene amplification and mutational activation of ACK1 are closely associated with human cancers. Particularly, the involvement of ACK1 in prostate cancer is well addressed. ACK1 phosphorylates the androgen receptor and the serine/threonine kinase Akt, leading to their activation. Overall, ACK1 is a key regulator of cell-surface receptor signaling in various types of cell responses including cytoskeletal rearrangements and transcriptional activation. Once excessively activated, ACK1 causes unregulated proliferation and survival, properties characteristic of cancer cells. Thus, ACK1 may be a promising therapeutic target of cancers.
- Linderoth E, Pilia G, Mahajan NP, Ferby I. Activated Cdc42-associated kinase 1 (Ack1) is required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor recruitment to lipid rafts and induction of cell death. J Biol Chem. 2013;288:32922–31.PubMedCentralCrossRefPubMedGoogle Scholar