RESUMEN
PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Proteína Quinasa C/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Endotelina-1/metabolismo , Activación Enzimática , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas con Dominio LIM/antagonistas & inhibidores , Proteínas con Dominio LIM/química , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM/antagonistas & inhibidores , Proteínas con Homeodominio LIM/química , Proteínas con Homeodominio LIM/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/química , Proteínas Musculares/genética , Miocitos Cardíacos/citología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteína Quinasa C/genética , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
We surveyed for covert baculovirus infections in the eastern spruce budworm, Choristoneura fumiferana (Clemens) and compared the prevalence of virus detected in a laboratory and a field population. DNA was extracted from budworm adults and then PCR with degenerate primers was used to identify individuals carrying baculovirus DNA. Multiplex PCR was then applied to the positive samples to distinguish between the multiple baculovirus types that could potentially be found in C. fumiferana populations. Covert infections were found in both the laboratory and the field population of C. fumiferana, although the frequency of infection and the composition of viruses found were very different. Overall 28% of insects from the laboratory population were positive for baculovirus DNA. Individual adults supported both single and mixed covert infections with CfMNPV plus CfDEFNPV, CfDEFNPV plus a GV and mixtures of all three viruses together. However, the majority of insects supported single virus infections, and surprisingly this virus was CfDEFNPV, a virus that is reported not to have per os activity in C. fumiferana larvae. Insects from field populations showed a very different pattern; 70.5% of individuals were baculovirus positive and all of these were positive for CfDEFNPV only.
