In addition to the previously identified Drosophila cdc2 and cdc2c genes, four additional cdc2-related genes have been identified with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologs of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologs is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appears to be ubiquitous according to in situ hybridization. Abundant expression already at the start of embryogenesis and long before neuron differentiation is also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice. Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions (Sauer, 1996).
Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. This study reports the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Rat Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Rat Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associates with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development (Choe, 2007; full text of article).
A Drosophila model with Mib1 loss-of-function was used to examine its physiological role and interaction with p35/CDK5. A P-element insertion was identified in the fly gene of CG5841, the Drosophila ortholog of Mib1. The homozygote was affirmed to be a mib1-null allele, because it showed pupal lethality that was rescued by precise removal of the transposon in the 5' untranslated region of the gene, or by the expression of transgenic mib1. The lack of full-length mib1 expression in the allele was verified by Western blotting. In Drosophila, the synaptic structure at the larval neuromuscular junction (NMJ) is a well defined system with which to study synaptic structure and neurotransmission, and the number of synaptic boutons is an established index for synaptic growth. By counting synaptic boutons in the wild-type and the mutant larvae, it was found that the loss of mib1 causes the synaptic overgrowth, and the number of synaptic boutons increased 85%. This finding suggests that Mib1 plays a conserved negative role in the formation of synaptic structure (Choe, 2007).
The genetic interaction between p35/CDK5 and mib1 was tested by monitoring adult eye phenotype. Overexpression of p35/CDK5 causes a rough eye phenotype, whereas the heterozygotic line of the mib1 mutant had a smooth eye phenotype. Crossing the p35/CDK5 transgenic line with the mib1 mutant line showed that the partial loss of mib1 in the heterozygote enhances the rough eye phenotype induced by p35/CDK5. This result also supports the negative regulation between p35/CDK5 and Mib1 in neuronal development (Choe, 2007).
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date revised: 1 November 2010
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