AKT Goes Cycling

AKT kinase is an important signaling molecule in multiple cell types, relaying cues of growth, proliferation, and survival ^1–3. AKT interacts with regulatory networks to feed extracellular signals into transcriptional programs within cells that eventually dictate the cell state. Its activation impacts a number of physiological processes such as glucose metabolism, protein synthesis and regulated apoptosis. In normal cells, activation of AKT kinase is thought to be critically dependent on its phosphorylations which are tightly regulated by disparate upstream kinases, responding to different stimuli ^4,5. Two of the best studied AKT phosphorylation events occur at threonine 308 and serine 473 residues, mediated by the receptor tyrosine kinase (RTK)/PI3K/PTEN signaling nexus ^6–10. The PI3K/PTEN pathway is one of the most deregulated pathways in human cancers. However, cancer cells often develop resistance to PI3K inhibitors or do not utilize the PI3K/PTEN pathway for AKT activation. Therefore, a number of laboratories have invested considerable efforts into understanding the mechanisms of AKT activation and its pathological role in driving human malignancies. Not surprisingly, these efforts have revealed that hyperactivation AKT in cancer cells is not solely mediated by the RTK/PI3K/PTEN signaling, but a diverse group of kinases could target and activate AKT to promote uncontrolled proliferation and resistance to chemotherapeutic agents ⁵ (Table 1). For example, some of the oncogenic kinases, such as ACK1 (also known as TNK2) and TBK1 were able to bypass the PI3K dependence to activate AKT and promote tumor growth and resistance to PI3K inhibitors ^4,11. Therefore, keeping in view the multiple regulatory networks that feed into AKT signaling and the complexity of signaling, pinpointing the mechanisms by which it is activated is crucial to specifically target this pathway to achieve maximum clinical benefit.

Table 1.

AKT phosphorylations and the corresponding kinases

Site of Phosphorylation	Domain	Kinase	References
Serine 137	Intradomain region between PH and Kinase domain	IKBKE	14,15
Tyrosine 176	Kinase domain	ACK1/TNK2	4,16
Theonine 195	Kinase domain	TBK1	11
Threonine 308	Kinase domain	PDK1	8
Tyrosine 315	Kinase domain	PTK6, SRC	17,18
Tyrosine 326	Kinase domain	PTK6, SRC	17,18
Serine 473	C-terminal region	mTORC2 complex	10
Serine 477/Threonine479	C-terminal region	Cdk2/Cyclin A	12

A recent report by Liu et. al. is one more addition in the already long list of AKT phosphorylations and highlights a previously unknown mode of AKT activation ¹². The authors have identified a novel phosphorylation event at the AKT carboxy terminus tail residues, Ser477/Thr479 that occurs in a cell cycle dependent manner. The key to this study is the generation of high specificity antibodies that cross react with the novel phosphoSer477/Thr479 AKT residues but not with phosphoserine473-AKT, which is a fairly robust phosphorylation ¹². Using a synchronized population of cycling cells, the authors have uncovered that not only does the AKT Ser477/Thr479-phosphorylation oscillate during the cell cycle, but it mirrors the periodic cyclin A2 expression and is catalyzed by the Cdk2/cyclin A2 complex, whose activity is itself regulated during the cell cycle. Importantly, the authors have identified four evolutionarily conserved RXL cyclin A-binding motifs in all the three human AKT isoforms as well as in the mouse and rat AKT. Mechanistically, the Cdk2/cyclin A2 mediated AKT phosphorylation at Ser477/Thr479 was found to enhance AKT activity by promoting the activating Ser473 phosphorylation. Accordingly, a phosphomimetic Akt1-DE mutant (Akt1-S477D/T479E), displayed increased S473 phosphorylation and had an enhanced ability to promote tumorigenesis in a mouse xenograft tumor model compared to the wild-type enzyme, while a double alanine mutant (Akt1-S477AD/T479A) showed loss of S473 phosphorylation, loss of substrate phosphorylation and decreased tumor development. Further, the authors have provided compelling evidence of Cdk2/Cyclin A2 complex as being the prime regulator of AKT Ser477/Thr479 phosphorylation by ectopically expressing Akt1-DE in mouse embryo fibroblasts (MEFs) derived from quadruple knockout KO mice (cyclin E1^−/−/cyclin E2^−/−/cyclin A1^−/−/cyclin A2^f/f) after transfection with Cre. Akt1-DE partly rescued the cell cycle defects observed in these MEFs.

At the molecular level, the Ser477/Thr479 phosphorylated AKT displayed increased association with Sin1 and mTOR complexes, but did not alter the association with phosphatases. Further, the authors have suggested that Ser477/Thr479 phosphorylation could lock the AKT kinase in an active conformation, a scenario that could also be observed if the carboxy-terminus of AKT is deleted. Interestingly, the Ser477 phosphorylation can still occur in a cell cycle independent manner, albeit not by Cdk2, but by the mTORC2/Rictor complex following insulin stimulation or during DNA damage by the related DNA-damage dependent protein kinase, DNA-PK. Further studies are required to understand the differential role of distinct kinases in AKT Ser477/Thr479 phosphorylation and its compartmentalization in cells.

What is the advantage of cell cycle specific regulation of AKT kinase activity? Is it an alternate mechanism to regulate AKT activation in cells to suppress untimely growth promoting signals? One can envisage a regulatory feedback loop wherein Cdk2 regulates cell cycle progression by acting on other substrates but also keeps the AKT signaling network under control by interacting with it and regulating AKT activity in a temporal manner and activated AKT in turn responds by transmitting growth signals that trigger re-entry into the cell cycle. Indeed, deletion of the Cyclin A2 in mouse embryonic stem cells impaired the AKT Ser477/Thr479 phosphorylation and caused elevated apoptosis. By the same token, it is possible to envisage the outcome when this regulation is lost in cancer cells. Liu et al also assessed this possibility and observed hyperphosphorylation of AKT at Ser477 in certain cancers ¹². For example, a positive correlation was observed between AKT-pSer477/pThr479 and pAKT-Ser473 in patients with breast cancer. However, in contrast to pSer473, high levels of pSer477/Thr479 occurred at a relatively higher rate in the earlier stages of breast cancer development. Whether this is indicative of a subset of rapidly cycling breast cells that are pre-disposed to overcome cell cycle checkpoints, develop genomic instability and become cancerous remains to be seen. If so, then as authors have suggested, this phosphorylation could be utilized as a biomarker to detect early stages of breast cancer.

With the identification of the novel mode of AKT phosphorylation, mediated by Cdk2/cyclin A2 complex, additional avenues to tackle tumor development became apparent. Cdk2, a crucial regulator of the cell division cycle, is overactive in many types of cancers and a number of inhibitors are now available to block its activity in cancer cells. One such inhibitor, Seliciclib (CYC202 or R-roscovitine) that inhibits CDK2, CDK7 and CDK9, has been evaluated in several Phase I and II studies and has shown early signs of anti-cancer activity. However, Cdk2 inhibitors may be not be completely effective in blocking cell cycle regulated AKT activation, as cancer cells may quickly adapt and employ mTORC2 pathways to compensate for the loss of Cdk2/Cyclin A activity. Therefore, direct AKT kinase inhibitors may be the key to target AKT that is being activated by multiple kinases. One such drug that appears to hold promise is an oral allosteric inhibitor of AKT, MK-2206, that is undergoing Phase II clinical trials ¹³. MK-2206 binds to the pleckstrin-homology (PH) domain of AKT and inhibits its activity in a non-ATP competitive manner by causing a change in conformation of AKT and preventing its localization to the plasma membrane.

Although, AKT phosphorylation at Ser477/Thr479 has been carefully examined for its cell cycle dependent modulation, how it is accomplished by Cdk2/cyclin A is far from clear. The majority of Cdk2 targets reside in the nucleus, so whether this phosphorylation occurs predominantly on nuclear AKT is not known. Interestingly, the phosphorylation was reduced in insulin stimulated cells treated with the PI3K inhibitor, LY29002, indicating that plasma membrane localization of AKT or the PI3K signaling has a role. However, the loss of AKT Ser477/Thr479 phosphorylation by LY29002 could be an indirect effect caused due to cell cycle arrest. Further, the relative amounts of AKT Ser477 phosphorylation as compared to Ser473 needs to be determined, which is critical to evaluate the contribution of this novel phosphorylation in the overall AKT activation.

In conclusion, normal as well as cancer cells appear to utilize the enzymatic activity of variety of kinases to maintain optimal AKT activity. Cdk2/Cyclin A2 mediated AKT phosphorylation adds up a new twist in this ongoing saga. However, unlike other AKT-interacting kinases (Table 1), Cdk2 is predominantly functional in nucleus and involved in regulation of G₁ to S phase progression of the cell cycle. Will this provide a new mode of tackling AKT activation as therapeutic strategy, remains to be seen.