Ack1-AKT signaling
The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). About a third of the breast and prostate tumors and majority of the pancreatic tumors that exhibit AKT activation, retain normal PTEN and PI3K activity. Interestingly, normal PTEN expression was also seen in breast, ovarian and prostate tumors that exhibit activated AKT. Overall, the molecular mechanisms regulating RTK mediated AKT activation in cancers with normal PTEN and PI3K activity is poorly understood.
We have uncovered a novel signaling mechanism wherein growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1. Ack1 directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain, a site that has not been reported in literature previously. Ack1 facilitates AKT plasma membrane localization; promotes Thr308/Ser473-phosphorylation leading to AKT activation. For membrane localization, AKT anchors to Phosphatidylinositol (3,4,5)-trisphosphate or PIP3, however, in contrast to AKT, Tyr176-phosphorylated AKT bound another membrane phospholipid, phosphatidic acid (PA). Collectively, our data indicates that a fraction of AKT that is Tyr176-phosphorylated can translocate to the membrane and undergo Ser473-phosphorylation and thus kinase activation even when PI3K is mutated/inactive.
Mahajan K et al. PLoS One. 2010;5(3):e9646
Detection of AKT Tyr176 by mass spectrometry. HA-tagged Tyr-phosphorylated AKT was purified followed by trypsin/chymotrypsin digestion. The peptide was detected at 13.83 mins in the total ion chromatogram with mass-to-charge ratio 647.8132, which represents an error of 0.38 ppm. The tandem mass spectrum matched the sequence, VKEKATGRYpY indicating that the C-terminal tyrosine was phosphorylated; the detection of the phosphotyrosine y1 is consistent with this localization.
The primary physiological outcome of Ack1 mediated AKT Tyr176-phosphorylation is the suppression of apoptotic genes expression and promotion of mitotic progression. This became amply evident in the mice expressing activated Ack1. We generated transgenic mice that specifically express Ack1 in prostate. These mice exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia or PINs.
The most significant role of pTyr176-AKT was observed in progression of breast cancer. The expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 or activated Ack1 were positively correlated with the severity of breast cancer progression, and inversely correlated with the survival of patients.
Large numbers of tumors are reliant upon AKT activation for survival and growth making it an attractive target for molecular therapeutics. Our data indicates that a new class of AKT inhibitors can be identified using antibodies against AKT Tyr176-phosphorylation (Contact us for pTyr176-AKT monoclonal antibodies). These novel inhibitors that block AKT membrane localization and activation could have major implications in cancer, diabetes and obesity research.
