Ack1 mediated AKT/PKB tyrosine 176 phosphorylation regulates its activation - PubMed (original) (raw)

. 2010 Mar 19;5(3):e9646.

doi: 10.1371/journal.pone.0009646.

Domenico Coppola, Sridevi Challa, Bin Fang, Y Ann Chen, Weiwei Zhu, Alexis S Lopez, John Koomen, Robert W Engelman, Charlene Rivera, Rebecca S Muraoka-Cook, Jin Q Cheng, Ernst Schönbrunn, Said M Sebti, H Shelton Earp, Nupam P Mahajan

Affiliations

Ack1 mediated AKT/PKB tyrosine 176 phosphorylation regulates its activation

Kiran Mahajan et al. PLoS One. 2010.

Abstract

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). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Tyr176 phosphorylation precedes AKT activation.

(A) MEF2KO cells were serum starved (24 h) and treated with EGF (10 ng/ml). The lysates were immunoprecipitated or IP with anti-Ack1 (top panel), anti-AKT (second panel) and anti-EGFR (fourth panel) antibodies followed by immunoblotting or IB with anti-pTyr antibodies. Remaining panel represents IB with antibodies as shown. (B) MEFs were serum starved (24 h) and treated with EGF (10 ng/ml for 10 mins) or pretreated with LY294002 (10 µM for 1 h) and EGF. The lysates were IP with Ack1 antibodies followed by IB with pan-AKT antibodies (top panel). (C) HA-tagged Tyr-phosphorylated AKT was purified (see Fig. S2A) followed by trypsin/chymotrypsin digestion. The peptide was detected at 13.83 mins in the total ion chromatogram (C) with mass-to-charge ratio 647.8132, which represents an error of 0.38 ppm (D). (E) 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. (F) Alignment of AKT protein sequences revealed that tyrosine at 176 is invariant from yeast to humans and all the three known human AKT isoforms. (G) MEF1&2KO cells expressing HA-tagged AKT or Y176F mutant were serum-starved (24 h), treated with EGF for 15 mins and lysates were IP with anti-Ack1 Abs followed by IB with anti-AKT antibodies (top panel). The lysates were also IP with anti-Ack1 antibodies followed by IB with pTyr antibodies (panel 4). The same blot was stripped and IB with anti-Ack1 antibodies (Bottom panel). These lysates were also subjected to IP with anti-HA antibodies followed by IB with Ser473, pTyr and AKT antibodies (panels 2, 3 and 5, respectively). (H) Flow cytometry of AKT and Y176F mutant expressing MEF1&2KO cells. Cells were serum starved for 24 h, treated with EGF for 15 mins, fixed and stained with HA-antibodies conjugated to Alexa488 and phosphoSer473-antibodies conjugated to Alexa 647. Upper right quadrant represents cells which express HA-tagged AKT or Y176F mutant that are also Ser473-phosphorylated.

Figure 2

Figure 2. Tyr176-phosphorylation regulates AKT plasma membrane localization.

(A) RWPE, normal prostate epithelial cells were treated with EGF (10 ng/ml,10 mins) and heregulin (10 ng/ml, 35 mins), whole cell protein lysates were subjected to IB with indicated antibodies. (B, C) MCF-7 cells were serum starved (24 h) and treated with (B) insulin (50 ng/ml) or (C) heregulin (30 ng/ml) for indicated times. Cell lysates were fractionated and IB with the indicated antibodies. Input panels pAck1(Tyr), pIR(Tyr) and pHER-2(Tyr) represents IP with respective antibodies followed by IB with pTyr antibodies. (D) MEF 1&2KO cells were transfected with HA-tagged AKT or Y176F mutant, serum starved (24 h) and treated with EGF for 15 mins. Cell lysates were fractionated and IB with anti-HA (top panel) and indicated antibodies (bottom panels). (E) MCF7 cells were transfected with control or Ack1-specific siRNAs (40 nM) for 48 h and treated with heregulin for 40 mins. Cell lysates were fractionated and IB with indicated antibodies. In this experiment we have used half the volumes buffer for extraction of cytosolic proteins. Thus, the cytosolic extracts are 2X concentrated as compared to Fig. 2B–C, which explains more p176-AKT in cytosol fraction than the plasma membrane fraction.

Figure 3

Figure 3. Tyr176-phosphorylation of AKT is PI3K-independent.

(A) MCF-7 cells were pretreated with LY294002 (10 µM, 1 h) followed by heregulin for 40 mins. Cell lysates were fractionated and membrane fraction was subjected to IB with indicated antibodies. (B) MCF-7 cells were mock transfected or transfected with control, Ack1 and PI3K siRNAs, followed by insulin treatment for 30 mins. Cell lysates were subjected to IP with pTyr-antibodies, followed by IB with pTyr176-AKT antibodies (top panel). Lower panels show IB with indicated antibodies. The experiment was performed with two different Ack1 siRNAs (Qiagen).

Figure 4

Figure 4. Tyr176 phosphorylated AKT suppresses FoxO gene transcription and promotes cell cycle progression.

(A) MEF1&2KO cells were transfected with caAck and HA-tagged AKT or Y176F, serum starved (24 h) and harvested. Total RNA was prepared and quantitative RT-PCR was performed. Data are representative of three independent experiments. *_p≤_0.05; **_p_≤0.03; ***_p_≤0.02; ****_p_≤0.02. (B) MEF2KO cells were transfected with control or Ack1-specific siRNAs (40 nM) for 48 h and treated with EGF for 30 mins. Total RNA was prepared and quantitative RT-PCR was performed. *_p_≤0.01; **_p_≤0.05; ***_p_≤0.06; ****_p≤_0.05. (C) Schematic representation of myr-AKT and myr-Y176F point mutants. SDM of myr-AKT was performed to generate the Y176F mutation. PH, Pleckstrin homology domain; Kinase, Kinase domain and CT, Carboxy Terminal regulatory region. (D) AKT MEF1&2 KO cells were transfected with HA-tagged myr-AKT or myr-Y176F mutant and harvested 24 h and 48 h post-transfection. Cells were fixed and stained with anti-HA antibodies conjugated with Alexa 488 and anti-pSerine10-Histone3 conjugated with Alexa 647, a marker used to distinguish cells in late G2 and early M phase, and analyzed by flow cytometry. HA-myrAKT expressing cells showed 75% increase in the number of cells undergoing mitosis (upper right quadrant), while, HA-myrY176F-AKT expressing mitotic cells remain unchanged.

Figure 5

Figure 5. Probasin-Ack1 transgenic mice display pTyr176-AKT and develop mPINs.

(A) Transgenic construct (Prob-Ack1) is shown. (B) A 25 wk old Probasin-Ack1 transgenic (TG) and wild type male mice prostate lysates were subjected to IP using anti-Myc antibodies followed by IB with pTyr antibodies (top panel). For bottom panels, lysates were subjected to IB with indicated antibodies. (C) Prostate lysates from 21 and 25 wk old TG and the WT siblings were IB with respective antibodies. The bottom 2 panels represent tail-PCR of these mice. IL-2 was an internal control for PCR. (D–L) Haematoxylin and eosin (H&E) stained WT and TG mice prostates. Histological appearance of the prostate lateral lobe from a 22 wk old WT mouse (D), and corresponding lobe from age-matched TG mice with intraepithelial hyperplasia (E). The lateral prostate from 49 wk old TG mice exhibiting mPIN (F) is shown. Contrasting histological appearance of the lateral, ventral and dorsal lobes of the prostate glands from a WT mouse (G–I), and corresponding lobes from TG mice (49 week old) are shown (J–L).

Figure 6

Figure 6. pTyr284-Ack1 and pTyr176-AKT expression in breast cancer.

(A) TMA sections representing different breast cancer stages stained with pTyr284-Ack1 and pTyr176-AKT antibodies. (B) Box plots to summarize distributions of staining intensities for pTyr284-Ack1 in different stages of breast cancer. A significant increasing trend of intensity across progression stages was detected (Mantel-Haenszel formula image test, p = 0.02). The box has lines at the lower quartile (25%), median (50%), and upper quartile values (75%) while the red-cross within the circle marks the mean value. Whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the ends of the box. The data with values beyond the ends of the whiskers, displayed with black circles, are potential outliers. (C) Box plots to summarize distributions of staining intensities for pTyr176-AKT in different stages of breast cancer. A significant increasing trend of intensity across progression stages was detected (Mantel-Haenszel formula image test, p<0.0001). (**D**) Kaplan–Meier analysis shows that individuals with breast cancer that have moderate to strong staining (>4) of pTyr284-Ack1 have a lower probability of survival (log rank test, p = 0.08). (E) Kaplan–Meier analysis of the breast cancer patients that have moderate to strong staining (>4) of pTyr176-AKT have significantly lower probability of survival (log rank test, p = 0.02).

Figure 7

Figure 7. Tyr176-phosphorylation leads to AKT activation, a model.

Our data demonstrates an alternate pathway of AKT activation wherein RTKs facilitate Ack1 phosphorylation at Tyr284 leading to its kinase activation. Ack1 could also be activated in some tumors by autoactivating somatic mutations, e.g. E346K. Activated Ack1 phosphorylates AKT at Tyr176 resulting in its binding to the anionic plasma membrane phospholipid PA. pTyr176-AKT localizes to the plasma membrane, where it is targeted by PDK1 and PDK2 (mTORC2 complex) for Thr308/Ser473 phosphorylations, respectively, leading to optimal AKT kinase activation. Activated AKT translocates to the nucleus, phosphorylates FoxO transcription factors to downregulate expression of FoxO target genes.

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