RESUMO
We used DNA microarray screening to identify Ckap2 (cytoskeleton associated protein 2) as a novel p53 target gene in a mouse erythroleukemia cell line. DNA damage induces human and mouse CKAP2 expression in a p53-dependent manner and p53 activates the Ckap2 promoter. Overexpressed Ckap2 colocalizes with and stabilizes microtubules. In p53-null cells, overexpression of Ckap2 induces tetraploidy with aberrant centrosome numbers, suggesting disturbed mitosis and cytokinesis. In p53-competent cells, Ckap2 does not induce tetraploidy but activates p53-mediated cell cycle arrest and apoptosis. Our data suggest the existence of a functional positive feedback loop in which Ckap2 activates the G1 tetraploidy checkpoint and prevents aneuploidy.
Assuntos
Aneuploidia , Proteínas do Citoesqueleto/genética , Leucemia Eritroblástica Aguda/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Ciclo Celular/genética , Centrossomo/fisiologia , Proteínas do Citoesqueleto/metabolismo , Células HCT116 , Humanos , Leucemia Eritroblástica Aguda/patologia , Camundongos , Microtúbulos/metabolismo , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Rho GTPases regulate reorganization of actin and microtubule cytoskeletal structures during both interphase and mitosis. The timing and subcellular compartment in which Rho GTPases are activated is controlled by the large family of Rho GTP exchange factors (RhoGEFs). Here, we show that the microtubule-associated RhoGEF Lfc is required for the formation of the mitotic spindle during prophase/prometaphase. The inability of cells to assemble a functioning spindle after Lfc inhibition resulted in a delay in mitosis and an accumulation of prometaphase cells. Inhibition of Lfc's primary target Rho GTPase during prophase/prometaphase, or expression of a catalytically inactive mutant of Lfc, also prevented normal spindle assembly and resulted in delays in mitotic progression. Coinjection of constitutively active Rho GTPase rescued the spindle defects caused by Lfc inhibition, suggesting the requirement of RhoGTP in regulating spindle assembly. Lastly, we implicate mDia1 as an important effector of Lfc signaling. These findings demonstrate a role for Lfc, Rho, and mDia1 during mitosis.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Prófase/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Animais , Linhagem Celular , Primers do DNA , Proteínas de Ligação ao GTP/genética , Vetores Genéticos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Microinjeções , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas/genética , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Carma1 (also known as caspase recruitment domain [CARD]11, Bimp3) is a CARD-containing membrane-associated guanylate kinase family protein that plays an essential role in antigen receptor-induced nuclear factor kappaB activation. We investigated the role of Carma1 in the assembly of signaling molecules at the immune synapse using a peptide-specific system. We report that Carma1 is essential for peptide-induced interleukin 2 and interferon gamma production, but dispensable for proliferation in T cells. Recruitment and distribution of T cell receptor, lymphocyte function associated 1, lipid rafts, and protein kinase C (PKC)theta; to central and peripheral immune synapse regions occur normally in Carma1-/- T cells. Carma1 controls entry of IkappaB kinase (IKK) into lipid raft aggregates and the central region of the immune synapse, as well as activation of IKK downstream of PKC. Our data provide the first genetic evidence on a new class of molecular scaffold that controls entry of defined signaling components, IKK, into the central supramolecular activation cluster at T cell-antigen-presenting cell interfaces without having any apparent effect on the overall organization and formation of immune synapses.
