UHRF2 regulates cell cycle, epigenetics and gene expression to control the timing of retinal progenitor and ganglion cell differentiation.

Ubiquitin-like, containing PHD and RING finger domains 2 (UHRF2) regulates cell cycle and binds 5-hydroxymethylcytosine (5hmC) to promote completion of DNA demethylation. Uhrf2-/- mice are without gross phenotypic defects; however, the cell cycle and epigenetic regulatory functions of Uhrf2 during retinal tissue development are unclear. Retinal progenitor cells (RPCs) produce all retinal neurons and Müller glia in a predictable sequence controlled by the complex interplay between extrinsic signaling, cell cycle, epigenetic changes and cell-specific transcription factor activation. In this study, we find that UHRF2 accumulates in RPCs, and its conditional deletion from mouse RPCs reduced 5hmC, altered gene expressions and disrupted retinal cell proliferation and differentiation. Retinal ganglion cells were overproduced in Uhrf2-deficient retinae at the expense of VSX2+ RPCs. Most other cell types were transiently delayed in differentiation. Expression of each member of the Tet3/Uhrf2/Tdg active demethylation pathway was reduced in Uhrf2-deficient retinae, consistent with locally reduced 5hmC in their gene bodies. This study highlights a novel role of UHRF2 in controlling the transition from RPCs to differentiated cell by regulating cell cycle, epigenetic and gene expression decisions.

Developmental Iron Deficiency Dysregulates TET Activity and DNA Hydroxymethylation in the Rat Hippocampus and Cerebellum.

Iron deficiency (ID) during neurodevelopment is associated with lasting cognitive and socioemotional deficits and increased risk for neuropsychiatric disease throughout the lifespan. These neurophenotypical changes are underlain by gene dysregulation in the brain that outlasts the period of ID; however, the mechanisms by which ID establishes and maintains gene expression changes are incompletely understood. The epigenetic modification of 5-hydroxymethylcytosine (5hmC), or DNA hydroxymethylation, is one candidate mechanism because of its dependence on iron-containing TET enzymes. The aim of the present study was to determine the effect of fetal-neonatal ID on regional brain TET activity, Tet expression, and 5hmC in the developing rat hippocampus and cerebellum and to determine whether changes are reversible with dietary iron treatment. Timed pregnant Sprague Dawley rats were fed iron-deficient diet (ID; 4 mg/kg Fe) from gestational day 2 to generate iron-deficient anemic (IDA) offspring. Control dams were fed iron-sufficient diet (IS; 200 mg/kg Fe). At postnatal day (P)7, a subset of ID-fed litters was randomized to IS diet, generating treated IDA (TIDA) offspring. At P15, the hippocampus and cerebellum were isolated for subsequent analysis. TET activity was quantified by ELISA from nuclear proteins. Expression of Tet1, Tet2, and Tet3 was quantified by qPCR from total RNA. Global %5hmC was quantified by ELISA from genomic DNA. ID increased DNA hydroxymethylation (p = 0.0105), with a corresponding increase in TET activity (p < 0.0001) and Tet3 expression (p < 0.0001) in the P15 hippocampus. In contrast, ID reduced TET activity (p = 0.0016) in the P15 cerebellum, with minimal effect on DNA hydroxymethylation. Neonatal dietary iron treatment resulted in partial normalization of these changes in both brain regions. These results demonstrate that the TET/DNA hydroxymethylation system is disrupted by developmental ID in a brain region-specific manner. Differential regional disruption of this epigenetic system may contribute to the lasting neural circuit dysfunction and neurobehavioral dysfunction associated with developmental ID.

Retinoblastoma tumor cell proliferation is negatively associated with an immune gene expression signature and increased immune cells.

This study focuses on gene expression differences between early retinal states that ultimately lead to normal development, late onset retinoblastoma, or rapid bilateral retinoblastoma tumors. The late-onset and early-onset retinoblastoma tumor cells are remarkably similar to normally proliferating retinal progenitor cells, but they fail to properly express differentiation markers associated with normal development. Further, early-onset retinoblastoma tumor cells express a robust immune gene expression signature followed by accumulation of dendritic, monocyte, macrophage, and T-lymphocyte cells in the retinoblastoma tumors. This characteristic was not shared by either normal retinae or late-onset retinoblastomas. Comparison of our data with other human and mouse retinoblastoma tumor gene expression significantly confirmed, that the immune signature is present in tumors from each species. Strikingly, we observed that the immune signature in both mouse and human tumors was most highly evident in those with the lowest proliferative capacity. We directly assessed this relationship in human retinoblastoma tumors by co-analyzing proliferation and immune cell recruitment by immunohistochemistry, uncovering a significant inverse relationship between increased immune-cell infiltration in tumors and reduced tumor cell proliferation. Directly inhibiting proliferation with a PI3K/mTOR inhibitor significantly increased the number of CD45+ immune cells in the retina. This work establishes an in vivo model for the rapid recruitment of immune cells to tumorigenic neural tissue.

An E2F1-dependent gene expression program that determines the balance between proliferation and cell death.

The Rb/E2F pathway regulates the expression of genes essential for cell proliferation but that also trigger apoptosis. During normal proliferation, PI3K/Akt signaling blocks E2F1-induced apoptosis, thus serving to balance proliferation and death. We now identify a subset of E2F1 target genes that are specifically repressed by PI3K/Akt signaling, thus distinguishing the E2F1 proliferative or apoptotic function. RNAi-mediated inhibition of several of these PI3K-repressed E2F1 target genes, including AMPK alpha 2, impairs apoptotic induction by E2F1. Activation of AMPK alpha 2 with an AMP analog further stimulates E2F1-induced apoptosis. We also show that the presence of the E2F1 apoptotic expression program in breast and ovarian tumors coincides with good prognosis, emphasizing the importance of the balance in the E2F1 proliferation/apoptotic program.

The nuclear protein UHRF2 is a direct target of the transcription factor E2F1 in the induction of apoptosis.

The E2F1 transcription factor is active in many types of solid tumors and can function as either an oncogene or tumor suppressor in vivo. E2F1 activity is connected with a variety of cell fates including proliferation, apoptosis, senescence, differentiation, and autophagy, and these effects are mediated through differential target gene expression. E2F1-induced cell death is an innate anti-cancer mechanism to kill cells with a spontaneous oncogenic mutation that might otherwise form a cancer. Relatively little is known about the molecular circuitry that tips E2F1 balance toward proliferation during normal growth versus apoptosis during oncogenic stress, and which pathways mediate this decision. To further explore these mechanisms, we utilized an unbiased shRNA screen to identify candidate genes that mediate E2F1-induced cell death. We identified the ubiquitin-like with PHD and ring finger domains 2 (UHRF2) gene as an important mediator of E2F1-induced cell death. UHRF2 encodes a nuclear protein involved in cell-cycle regulation. Several of these domains have been shown to be essential for the regulation of cell proliferation, and UHRF2 has been implicated as an oncogene in some settings. Other reports have suggested that UHRF2 causes growth arrest, functions as a tumor suppressor, and is deleted in a variety of tumors. We show that UHRF2 is a transcriptional target of E2F, that it directly interacts with E2F1, and is required for E2F1 induction of apoptosis and transcription of a number of important apoptotic regulators.

UHRF2 accumulates in early G1-phase after serum stimulation or mitotic exit to extend G1 and total cell cycle length.

Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can't perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.

Early-Life Iron Deficiency Anemia Programs the Hippocampal Epigenomic Landscape.

Iron deficiency (ID) anemia is the foremost micronutrient deficiency worldwide, affecting around 40% of pregnant women and young children. ID during the prenatal and early postnatal periods has a pronounced effect on neurodevelopment, resulting in long-term effects such as cognitive impairment and increased risk for neuropsychiatric disorders. Treatment of ID has been complicated as it does not always resolve the long-lasting neurodevelopmental deficits. In animal models, developmental ID results in abnormal hippocampal structure and function associated with dysregulation of genes involved in neurotransmission and synaptic plasticity. Dysregulation of these genes is a likely proximate cause of the life-long deficits that follow developmental ID. However, a direct functional link between iron and gene dysregulation has yet to be elucidated. Iron-dependent epigenetic modifications are one mechanism by which ID could alter gene expression across the lifespan. The jumonji and AT-rich interaction domain-containing (JARID) protein and the Ten-Eleven Translocation (TET) proteins are two families of iron-dependent epigenetic modifiers that play critical roles during neural development by establishing proper gene regulation during critical periods of brain development. Therefore, JARIDs and TETs can contribute to the iron-mediated epigenetic mechanisms by which early-life ID directly causes stable changes in gene regulation across the life span.

Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor.

Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase alpha together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.

Retinoblastoma cells activate the AKT pathway and are vulnerable to the PI3K/mTOR inhibitor NVP-BEZ235.

Retinoblastoma is a pediatric cancer of the retina most often caused by inactivation of the retinoblastoma (RB1) tumor suppressor gene. We previously showed that Rb1 loss cooperates with either co-activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, or co-deleting Pten, to initiate retinoblastoma tumors in mice. The objectives of this study were to determine if the AKT pathway is activated in human retinoblastomas and the extent that anti-PI3K therapy induces apoptosis in retinoblastoma cells, alone or in combination with the DNA damaging drugs carboplatin and topotecan. Serial sections from human retinoblastoma tissue microarrays containing 27 tumors were stained with antibodies specific to p-AKT, Ki-67, forkhead box O1 (p-FOXO1), and ribosomal protein S6 (p-S6) using immunohistochemistry and each tumor sample scored for intensity. Human retinoblastoma tumors displayed significant correlation between p-AKT intensity with highly proliferative tumors (p = 0.008) that were also highly positive for p-FOXO1 (p = 0.002). Treatment with BEZ235, a dual PI3K/mTOR inhibitor, reduced phosphorylation levels of the AKT targets p-FOXO and p-S6 and effectively induced apoptosis the Y79 and Weri-1 human retinoblastoma cell lines and in vivo in our retinoblastoma mouse model. Long-term treatment with BEZ235 in vivo using our retinoblastoma-bearing mice induced apoptosis but did not significantly extend the lifespan of the mice. We then co-administered BEZ235 with topotecan and carboplatin chemotherapeutics in vivo, which more effectively induced apoptosis of retinoblastoma, but not normal retinal cells than either treatment alone. Our study has increased the variety of potentially effective targeted treatments that can be considered for human retinoblastoma.

Loss of UHRF2 expression is associated with human neoplasia, promoter hypermethylation, decreased 5-hydroxymethylcytosine, and high proliferative activity.

Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) binds to 5-hydroxymethylcytosine (5hmC), a DNA base involved in tissue development, but it is unknown how their distribution compares with each other in normal and malignant human tissues. We used IHC on human tumor specimens (160 from 19 tumor types) or normal tissue to determine the expression and distribution of UHRF2, Ki-67, and 5hmC. We also examined UHRF2 expression in cord blood progenitors and compared its expression to methylation status in 6 leukemia cell lines and 15 primary human leukemias. UHRF2 is highly expressed, paralleling that of 5hmC, in most non-neoplastic, differentiated tissue with low Ki-67 defined proliferative activity. UHRF2 is expressed in common lymphoid progenitors and mature lymphocytes but not common myeloid progenitors or monocytes. In contrast, UHRF2 immunostaining in human cancer tissues revealed widespread reduction or abnormal cytoplasmic localization which correlated with a higher Ki-67 and reduced 5hmC. UHRF2 expression is reduced in some leukemia cell lines, this correlates with promoter hypermethylation, and similar UHRF2 methylation profiles are seen in primary human leukemia samples. Thus, UHRF2 and 5hmC are widely present in differentiated human tissues, and UHRF2 protein is poorly expressed or mislocalized in diverse human cancers.

Rb1 and Pten Co-Deletion in Osteoblast Precursor Cells Causes Rapid Lipoma Formation in Mice.

The Rb and Pten tumor suppressor genes are important regulators of bone development and both are frequently mutated in the bone cancer osteosarcoma (OS). To determine if Rb1 and Pten synergize as tumor suppressor genes for osteosarcoma, we co-deleted them in osteoprogenitor cells. Surprisingly, we observed rapid development of adipogenic but not osteosarcoma tumors in the ΔRb1/Pten mice. ΔPten solo deleted mice also developed lipoma tumors but at a much reduced frequency and later onset than those co-deleted for Rb1. Pten deletion also led to a marked increase in adipocytes in the bone marrow. To better understand the function of Pten in bone development in vivo, we conditionally deleted Pten in OSX(+) osteoprogenitor cells using OSX-Cre mice. µCT analysis revealed a significant thickening of the calvaria and an increase in trabeculae volume and number in the femur, consistent with increased bone formation in these mice. To determine if Pten and Rb1 deletion actively promotes adipogenic differentiation, we isolated calvarial cells from Pten(fl/fl) and Pten(fl/fl); Rb1(fl/fl) mice, infected them with CRE or GFP expressing adenovirus, treated with differentiation media. We observed slightly increased adipogenic, and osteogenic differentiation in the ΔPten cells. Both phenotypes were greatly increased upon Rb1/Pten co-deletion. This was accompanied by an increase in expression of genes required for adipogenesis. These data indicate that Pten deletion in osteoblast precursors is sufficient to promote frequent adipogenic, but only rare osteogenic tumors. Rb1 hetero- or homo-zygous co-deletion greatly increases the incidence and the rapidity of onset of adipogenic tumors, again, with only rare osteosarcoma tumors.

Sensitivity to TOP2 targeting chemotherapeutics is regulated by Oct1 and FILIP1L.

Topoisomerase II (TOP2) targeting drugs like doxorubicin and etoposide are frontline chemotherapeutics for a wide variety of solid and hematological malignancies, including breast and ovarian adenocarcinomas, lung cancers, soft tissue sarcomas, leukemias and lymphomas. These agents cause a block in DNA replication leading to a pronounced DNA damage response and initiation of apoptotic programs. Resistance to these agents is common, however, and elucidation of the mechanisms causing resistance to therapy could shed light on strategies to reduce the frequency of ineffective treatments. To explore these mechanisms, we utilized an unbiased shRNA screen to identify genes that regulate cell death in response to doxorubicin treatment. We identified the Filamin A interacting protein 1-like (FILIP1L) gene as a crucial mediator of apoptosis triggered by doxorubicin. FILIP1L shares significant similarity with bacterial SbcC, an ATPase involved in DNA repair. FILIP1L was originally described as DOC1, or "down-regulated in ovarian cancer" and has since been shown to be downregulated in a wide variety of human tumors. FILIP1L levels increase markedly through transcriptional mechanisms following treatment with doxorubicin and other TOP2 poisons, including etoposide and mitoxantrone, but not by the TOP2 catalytic inhibitors merbarone or dexrazoxane (ICRF187), or by UV irradiation. This induction requires the action of the OCT1 transcription factor, which relocalizes to the FILIP1L promoter and facilitates its expression following doxorubicin treatment. Our findings suggest that the FILIP1L expression status in tumors may influence the response to anti-TOP2 chemotherapeutics.

Jab1/CSN5 mediates E2F dependent expression of mitotic and apoptotic but not DNA replication targets.

The E2F transcription factors are critical regulators of cell cycle and cell fate control. Several classes of E2F target genes have been categorized based on their roles in DNA replication, mitosis, apoptosis, DNA repair, etc. How E2Fs coordinate the appropriate and timely expression of these functionally disparate gene products is poorly understood at a molecular level. We previously showed that the E2F1 binding partner Jab1/CSN5 promotes E2F1-dependent induction of apoptosis but not proliferation. To better understand how Jab1 regulates E2F1 dependent transcription, we performed gene expression analysis to identify E2F target genes most and least affected by shRNA depletion of Jab1. We find that a significant number of apoptotic and mitotic E2F target genes are poorly expressed in cells lacking Jab1/CSN5, whereas DNA replication genes are generally still highly expressed. Chromatin immunoprecipitation analysis indicates that both Jab1 and E2F1 co-occupy apoptotic and mitotic, but not DNA replication target genes. We explored a potential connection between PI3K activity and Jab1/E2F1 target gene induction, and found that E2F1/Jab1 co-induction of apoptotic target genes can be inhibited by activated PI3K. Furthermore, PI3K activity interferes with formation of the E2F1/Jab1 complex by co-immunoprecipitation. Jab1/CSN5 is upregulated in a variety of human tumors, but it's unclear how its pro-proliferatory and apoptotic functions are regulated in this context. We explored the link between increased Jab1 levels and PI3K function in tumors and detected a highly significant correlation between elevated Jab1/CSN5 levels and PI3K activity in breast, ovarian, lung and prostate cancers.

Androgens suppress EZH2 expression via retinoblastoma (RB) and p130-dependent pathways: a potential mechanism of androgen-refractory progression of prostate cancer.

Androgens and the androgen receptor are important for both normal prostate development and progression of prostate cancer (PCa). However, the underlying mechanisms are not fully understood. The Polycomb protein enhancer of zeste homolog 2 (EZH2) functions as an epigenetic gene silencer and plays a role in oncogenesis by promoting cell proliferation and invasion. EZH2 has been implicated in human PCa progression, because its expression is often elevated in hormone-refractory PCa. Here, we demonstrated that expression of EZH2 is lower in androgen-sensitive LNCaP PCa cells compared with Rf and C4-2 cells, two androgen-refractory sublines that are derived from LNCaP cells. Androgen ablation by castration increased the level of EZH2 proteins in LNCaP xenografts in mice. In contrast, treatment of LNCaP cells in culture with the synthetic androgen methyltrieolone (R1881) at doses of 1 nm or higher suppressed EZH2 expression. Moreover, our data suggest that androgen repression of EZH2 requires a functional androgen receptor and this effect is mediated through the retinoblastoma protein and its related protein p130. We further showed that androgen treatment not only increases expression of EZH2 target genes DAB2IP and E-cadherin but also affects LNCaP cell migration. Our results reveal that androgens function as an epigenetic regulator in prostatic cells by repression of EZH2 expression through the retinoblastoma protein and p130-dependent pathways. Our findings also suggest that blockade of EZH2 derepression during androgen deprivation therapy may represent an effective tactic for the treatment of androgen-refractory PCa.

Balancing the decision of cell proliferation and cell fate.

The control of cellular proliferation is key in the proper development of a complex organism, the maintenance of tissue homeostasis and the ability to respond to various hormonal and other inducers. Key in the control of proliferation is the retinoblastoma (Rb) protein which regulates the activity of a family of transcription factors known as E2Fs. The E2F proteins are now recognized to regulate the expression of a large number of genes associated with cell proliferation including genes encoding DNA replication as well as mitotic activities. What has also become clear over the past several years is the intimate relationship between the control of cell proliferation and the control of cell fate, particularly the activation of apoptotic pathways. Central in this connection is the Rb/E2F pathway that not only provides the primary signals for proliferation but at the same time, connects with the p53-dependent apoptotic pathway. This review addresses this inter-connection and the molecular mechanisms that control the decision between proliferation and cell death.

Jab1 is a specificity factor for E2F1-induced apoptosis.

The members of the E2F family of transcription factors are key regulators of genes involved in cell cycle progression, cell fate determination, DNA damage repair, and apoptosis. Many cell-based experiments suggest that E2F1 is a stronger inducer of apoptosis than the other E2Fs. Our previous work identified the E2F1 marked box and flanking region as critical for the specificity in E2F1 apoptosis induction. We have now used a yeast two-hybrid screen to identify proteins that bind the E2F1 marked box and flanking regions, with a potential role in E2F1 apoptosis induction. We identified Jab1 as an E2F1-specific binding protein and showed that Jab1 and E2F1 coexpression synergistically induce apoptosis, coincident with an induction of p53 protein accumulation. In contrast, Jab1 does not synergize with E2F1 to promote cell cycle entry. Cells depleted of Jab1 are deficient for both E2F1-induced apoptosis and induction of p53 accumulation. We suggest that Jab1 is an essential cofactor for the apoptotic function of E2F1.

Transcriptional regulation by Lge1p requires a function independent of its role in histone H2B ubiquitination.

Saccharomyces cerevisiae cells that have lost their mitochondrial genome (rho(0)) strongly induce transcription of multidrug resistance genes, including the ATP-binding cassette transporter gene PDR5. PDR5 induction in rho(0) cells requires the presence of the zinc cluster transcription factor Pdr3p. The PDR3 gene is positively autoregulated in rho(0) cells by virtue of the presence of two binding sites for Pdr3p in its promoter. We identify the novel protein Lge1p as a required participant in the rho(0) activation of PDR3 and PDR5 expression. Lge1p is a nuclear protein that has been found to play a role in ubiquitination of histone H2B at Lys(123). This ubiquitination requires the presence of the ubiquitin-conjugating enzyme Rad6p and the ubiquitin ligase Bre1p. Interestingly, rho(0) strains lacking Lge1p failed to induce PDR3 transcription, but induction was still seen in Deltarad6, Deltabre1, and H2B-K123R mutant strains. Microarray experiments also confirmed that the pattern of gene expression changes seen in cells lacking Lge1p, Bre1p, or Rad6p or containing the H2B-K123R mutant as the only form of H2B share some overlap but are distinct. These findings provide a strong argument that Lge1p has roles in gene regulation independent of its participation in the Rad6p-dependent ubiquitination of H2B.

Specificity in the activation and control of transcription factor E2F-dependent apoptosis.

Previous work has demonstrated a role for the E2F1 gene product in signaling apoptosis, both as a result of the deregulation of the Rb/E2F pathway as well as in response to DNA damage. We now show that the ability of cells to suppress the apoptotic potential of E2F1, as might occur during the course of normal cellular proliferation, requires the action of the Ras-phosphoinositide 3-kinase-Akt signaling pathway. In addition, we also identify a domain within the E2F1 protein, previously termed the marked-box domain, that is essential for the apoptotic activity of E2F1 and that distinguishes the E2F1 protein from E2F3. We also show that the E2F1-marked-box domain is essential for the induction of both p53 and p73 accumulation. Importantly, a role for the marked-box domain in the specificity of E2F1-mediated apoptosis coincides with recent work demonstrating a role for this domain in achieving specificity in the activation of transcription. We conclude that the unique capacity of E2F1 to trigger apoptosis reflects a specificity of transcriptional activation potential, and that this role for E2F1 is regulated through the action of the Akt protein kinase.