RESUMEN
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an unstable (CTG . CAG)n segment in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. It is commonly accepted that DMPK mRNA-based toxicity is the main contributor to DM1 manifestations; however, not much is known about the significance of the DMPK protein. To appreciate its normal and possible pathobiological role, we analyzed the patterns of DMPK splice isoform expression in mouse tissues. Long membrane-anchored DMPK dominated in heart, diaphragm, and skeletal muscle, whereas short cytosolic isoforms were highly expressed in bladder and stomach. Both isoform types were present in diverse brain regions. DMPK protein was also detectable in cultured myoblasts, myotubes, cortical astrocytes, and related cell lines of neural or muscle origin, but not in hippocampal neurons. This work identifies DMPK as a kinase with pronounced expression in diverse muscle and neural tissues that are affected in DM1.
Asunto(s)
Linaje de la Célula/fisiología , Células Musculares/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Astrocitos/metabolismo , Western Blotting , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Inmunoprecipitación , Isomerismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A', and the brain specific Rab6B. Recent studies have shown that Rab6A' is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediate chains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A' and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity.
Asunto(s)
Dineínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Dineínas Citoplasmáticas , Dineínas/química , Dineínas/ultraestructura , GTP Fosfohidrolasas/metabolismo , Aparato de Golgi/ultraestructura , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas de Unión al GTP rab/químicaRESUMEN
The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neuronas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Células COS , Chlorocebus aethiops , Vesículas Citoplasmáticas/metabolismo , Proteínas del Citoesqueleto/química , Aparato de Golgi/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
The single-copy mouse gene Ptprr gives rise to different protein tyrosine phosphatase (PTP) isoforms in neuronal cells through the use of distinct promoters, alternative splicing, and multiple translation initiation sites. Here, we examined the array of post-translational modifications imposed on the PTPRR protein isoforms PTPBR7, PTP-SL, PTPPBSgamma42 and PTPPBSgamma37, which have distinct N-terminal segments and localize to different parts of the cell. All isoforms were found to be short-lived, constitutively phosphorylated proteins. In addition, the transmembrane isoform, PTPBR7, was subject to N-terminal proteolytic processing, in between amino acid position 136 and 137, resulting in an additional, 65-kDa transmembrane PTPRR isoform. Unlike for some other receptor-type PTPs, the proteolytically produced N-terminal ectodomain does not remain associated with this PTPRR-65. Shedding of PTPBR7-derived polypeptides at the cell surface further adds to the molecular complexity of PTPRR biology.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Encéfalo/metabolismo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas Clase 7 Similares a Receptores , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The use of alternative splice sites, promoters and translation start sites considerably adds to the complexity of organisms. Four mouse cDNAs (PTPBR7, PTP-SL, PTPPBSgamma+ and PTPPBSgamma-) have been cloned that contain different 5' parts but encode identical protein tyrosine phosphatase PTPRR catalytic domains. We investigated the genomic origin and coding potential of these transcripts to elucidate their interrelationship. Mouse gene Ptprr exons were identified within a 260 kbp segment on chromosome 10, revealing PTP-SL- and PTPPBSgamma-specific transcription start sites within introns two and four, respectively, relative to the 14 PTPBR7 exons. Northern and RT-PCR analyses demonstrated differential expression patterns for these promoters. Furthermore, transfection studies and AUG codon mutagenesis demonstrated that in PTP-SL and PTPPBSgamma messengers multiple translation initiation sites are being used. Resulting 72, 60, 42 and 37 kDa PTPRR protein isoforms differ not only in the length of their N-terminal part but also in their subcellular localization, covering all major PTP subtypes; receptor-like, membrane associated and cytosolic. In summary, mouse gene Ptprr gives rise to multiple isoforms through the use of distinct promoters, alternative splicing and differential translation starts. These results set the stage for further investigations on the physiological roles of PTPRR proteins.