RESUMEN
Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.
Asunto(s)
Ciclo Celular , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Simulación de Dinámica Molecular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/química , Humanos , Unión Proteica , Ciclina E/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina D/metabolismo , Ciclina D/química , Ciclina D/genética , Sitios de Unión , Activación EnzimáticaRESUMEN
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.
Asunto(s)
Proliferación Celular/genética , Ciclina D/genética , Mapas de Interacción de Proteínas/genética , Proteína de Retinoblastoma/genética , Ciclo Celular/genética , Proteína Sustrato Asociada a CrK/genética , Ciclina D/química , Quinasa 4 Dependiente de la Ciclina/química , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/química , Quinasa 6 Dependiente de la Ciclina/genética , Ciclinas/genética , Fase G1/genética , Humanos , Simulación del Acoplamiento Molecular , Fosforilación/genética , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Proteína de Retinoblastoma/química , Proteína p107 Similar a la del Retinoblastoma/genética , Fase S/genéticaRESUMEN
Understanding the intricate steps of protein kinase regulation requires characterization of protein-protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP-2Rho, to peptide and protein derivatives of cyclin-dependent kinaseâ 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclinâ D1 and a peptide substrate. We demonstrate the sensitivity of TP-2Rho in relation to of those other dyes used for monitoring peptide-protein and protein-protein interactions. Moreover, we show that TP-Rho-labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclinâ D.
Asunto(s)
Ciclina D/química , Ciclina D/metabolismo , Quinasa 4 Dependiente de la Ciclina/química , Quinasa 4 Dependiente de la Ciclina/metabolismo , Colorantes Fluorescentes/química , Maleimidas/química , Tiazolidinas/química , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Maleimidas/síntesis química , Modelos Moleculares , Estructura Molecular , Unión Proteica , Tiazolidinas/síntesis químicaRESUMEN
Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides - a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication.
Asunto(s)
Gammaherpesvirinae/fisiología , Interacciones Huésped-Patógeno , Modelos Biológicos , Fosfoproteínas/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Replicación Viral , Células 3T3 , Animales , Cromatografía Líquida de Alta Presión , Biología Computacional , Ciclina D/química , Ciclina D/genética , Ciclina D/metabolismo , Gammaherpesvirinae/genética , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Proteoma/química , Proteoma/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/metabolismo , Espectrometría de Masas en Tándem , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FST > AGN > CHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme.
Asunto(s)
Apigenina/química , Ciclina D/química , Quinasa 6 Dependiente de la Ciclina/química , Flavonoides/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Apigenina/farmacología , Sitios de Unión , Cristalografía por Rayos X , Ciclina D/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Flavonoides/farmacología , Flavonoles , Humanos , Enlace de Hidrógeno , Conformación Molecular , Simulación de Dinámica Molecular , Piperidinas/química , Piperidinas/farmacología , Unión Proteica , Electricidad Estática , TermodinámicaRESUMEN
p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.
Asunto(s)
Ciclina D/química , Quinasa 4 Dependiente de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/química , Complejos Multiproteicos/química , Pliegue de Proteína , Ciclina D/genética , Ciclina D/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Entropía , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismoRESUMEN
Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins.