RESUMEN
Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.
Asunto(s)
Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Células HCT116 , Humanos , Fosforilación , Dominios Homólogos a Pleckstrina , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-akt/química , Serina/química , Transducción de Señal , Treonina/químicaRESUMEN
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.
Asunto(s)
Dominios Homólogos a Pleckstrina , Proteínas Proto-Oncogénicas c-akt , Oncogenes , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genéticaRESUMEN
Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt's kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células HCT116 , Humanos , Insectos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/síntesis química , Células Sf9RESUMEN
Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here, we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/química , Línea Celular , Clonación Molecular , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación , Conformación Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Relación Estructura-ActividadRESUMEN
Akt1-3 (Akt) are a subset of the AGC protein Ser/Thr kinase family and play important roles in cell growth, metabolic regulation, cancer, and other diseases. We describe some of the roles of Akt in cell signaling and the biochemical and structural mechanisms of the regulation of Akt catalysis by the phospholipid PIP3 and by phosphorylation. Recent findings highlight a diverse set of strategies to control Akt catalytic activity to ensure its normal biological functions.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Catálisis , Humanos , Fosfatidilinositol 3-Quinasas , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (â¼ 4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.
Asunto(s)
Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/metabolismo , Lisina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Acetilación , Anhidrasa Carbónica II/genética , Escherichia coli/genética , Humanos , Lisina/química , Modelos Moleculares , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteínas Recombinantes/genéticaRESUMEN
Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.