RESUMO
Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an "in situ" approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5'-triphosphate analogs. We identified 117 candidate substrates, ~40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.
RESUMO
Fungal infections of the central nervous system (CNS) are associated with high mortality rates in immunocompromised patients. Surgical intervention is a mainstay of therapy, but not always possible. We describe the use of medical therapy for the treatment of CNS fungal infections in four pediatric cancer patients. Definitive resection was not performed in any patient. All patients initially received combination antifungal therapy with good clinical response; long-term survival was documented in two patients able to transition to long-term azole therapy. Prolonged antifungal therapy is an important option for treating invasive CNS fungal infections when surgery is not feasible.
Assuntos
Antifúngicos/uso terapêutico , Infecções do Sistema Nervoso Central/tratamento farmacológico , Micoses/tratamento farmacológico , Neoplasias/complicações , Adolescente , Adulto , Infecções do Sistema Nervoso Central/mortalidade , Criança , Feminino , Humanos , Masculino , Micoses/mortalidadeRESUMO
Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.
Assuntos
Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Mitose , Dados de Sequência Molecular , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Fosforilação , Mutação Puntual , Serina/química , Serina/genética , Treonina/química , Treonina/metabolismoRESUMO
The early growth response (Egr) family of transcriptional regulators consists of four proteins that share highly conserved DNA-binding domains. In many cell types, they are coexpressed and appear to have cooperative roles in regulating gene expression during growth and differentiation. Three Egr proteins, Egr1, Egr2, and Egr3, are induced during thymocyte differentiation in response to pre-TCR signaling, suggesting they may be critical for some aspects of pre-TCR-mediated differentiation. Indeed, enforced expression of Egr proteins in developing thymocytes can recapitulate some aspects of pre-TCR signaling, but the mechanisms by which they contribute to beta-selection are still poorly understood. Egr3 stimulates proliferation of beta-selected thymocytes, and Egr3-deficient mice have hypocellular thymuses, defects in proliferation, and impaired progression from double-negative 3 to double-negative 4. Surprisingly, Egr1-deficient mice exhibit normal beta-selection, indicating that the functions of Egr1 during beta-selection are likely compensated by other Egr proteins. In this study, we show that mice lacking both Egr1 and Egr3 exhibit a more severe thymic atrophy and impairment of thymocyte differentiation than mice lacking either Egr1 or Egr3. This is due to a proliferation defect and cell-autonomous increase in apoptosis, indicating that Egr1 and Egr3 cooperate to promote thymocyte survival. Microarray analysis of deregulated gene expression in immature thymocytes lacking both Egr1 and Egr3 revealed a previously unknown role for Egr proteins in the maintenance of cellular metabolism, providing new insight into the function of these molecules during T cell development.
Assuntos
Diferenciação Celular/imunologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Proteína 3 de Resposta de Crescimento Precoce/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Atrofia , Diferenciação Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/deficiência , Proteína 3 de Resposta de Crescimento Precoce/biossíntese , Proteína 3 de Resposta de Crescimento Precoce/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Timo/metabolismo , Timo/patologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Early growth response (Egr) proteins comprise a family of transcriptional regulators (Egr1-4) that modulate gene expression involved in the growth and differentiation of many cell types. In particular, Egr1 is widely believed to have an essential role in regulating monocyte/macrophage differentiation. However, Egr1-deficient mice have normal numbers of functional macrophages, an observation that has led to the hypothesis that other Egr proteins may compensate for Egr1 function in vivo. We examined whether other Egr transcription factors have a functionally redundant role in monocyte/macrophage differentiation. Egr1 and Egr3 expression was found to be induced in myeloid cells when they were differentiated into macrophages by treatment with M-CSF, whereas Egr2 was minimally induced and Egr4 was not detected. In either Egr1/Egr3 or Egr1/Egr2 double homozygous mutant mice, macrophage differentiation and function remained unimpaired. Additionally, the expression of molecules that broadly inhibit Egr function failed to block commitment to the monocytic lineage or inhibit the maturation of monocyte precursors. Finally, several hemopoietic growth factors were found to induce Egr gene expression, indicating that Egr gene expression is not cell lineage specific. Taken together, these results demonstrate that Egr transcription factors are neither essential for nor specific to monocyte/macrophage differentiation.