RESUMO
In the kinesin family, all the molecular motors that have been implicated in the regulation of microtubule dynamics have been shown to stimulate microtubule depolymerization. Here, we report that kinesin-1 (also known as conventional kinesin or KIF5B) stimulates microtubule elongation and rescues. We show that microtubule-associated kinesin-1 carries the c-Jun N-terminal kinase (JNK) to allow its activation and that microtubule elongation requires JNK activity throughout the microtubule life cycle. We also show that kinesin-1 and JNK promoted microtubule rescues to similar extents. Stimulation of microtubule rescues by the kinesin-1/JNK pathway could not be accounted for by the rescue factor CLIP-170. Indeed only a dual inhibition of kinesin-1/JNK and CLIP-170 completely blocked rescues and led to extensive microtubule loss. We propose that the kinesin-1/JNK signaling pathway is a major regulator of microtubule dynamics in living cells and that it is required with the rescue factor CLIP-170 to allow cells to build their interphase microtubule network.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Western Blotting , Imunofluorescência , Genes Dominantes , Células HeLa , Humanos , Imunoprecipitação , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Microinjeções , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fuso AcromáticoRESUMO
Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.
Assuntos
Guanosina Trifosfato/análise , Microtúbulos/química , Tubulina (Proteína)/química , Animais , Anticorpos/imunologia , Linhagem Celular , Simulação por Computador , Dimerização , Imunofluorescência , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Método de Monte Carlo , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Tubulina (Proteína)/análise , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/metabolismoRESUMO
We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.