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Please use this identifier to cite or link to this item: http://hdl.handle.net/10277/255

Authors: Giudici, Laura
Title: The c-terminal tail and the catalytic activity of cdkl5 are fine regulators of its nucleo-cytoplasmic shuttling.
Abstract: Rett syndrome (RTT) is an X-linked neurological disorder affecting mainly females. In the classical form, patients have a normal period of development of 6-18 months whereafter they display developmental arrest and a progressive regression leading to the loss of speech and purposeful movements with the appearance of a severe mental retardation. Several RTT variants have been described ranging from milder forms with a later age of onset to more severe manifestations. Mutations in the methyl-CpG-binding protein (MECP2) gene, located on Xq28, cause the majority of RTT cases but have been found in less than 50% of patients affected by the variant forms. Recently, mutations in the cyclindependent kinase-like 5 (CDKL5) gene, on Xp22, have been found in some RTT patients with the Hanefeld variant, characterized by the onset of seizures in the very first months of life. Furthermore, mutations in CDKL5 have been found in girls with infantile spasms and mental retardation, suggesting an important role of this gene for neuronal function. Nevertheless, the role of this kinase in the nervous system remain almost completely uncharacterized. We are characterizing the role of CDKL5 in the nervous system in order to clarify the molecular mechanisms involved in disease onset. We have previously shown that CDKL5 and MeCP2 function in a common pathway in accordance with the fact that mutations in the two genes cause a similar phenotype. In fact, besides sharing an overlapping expression pattern, correlating with neuronal maturation and synaptogenesis, CDKL5 and MeCP2 associate and in vitro the kinase is capable of mediating the phosphorylation of the methyl-CpG-binding protein. These results have led to the current model assuming that CDKL5 works upstream of MeCP2 and influences directly or indirectly its phosphorylation and functions. Albeit CDKL5 remains largely uncharacterized, our experiments in cultured cells show that CDKL5 shuttles between the nucleus and the cytoplasm and that an active nuclear export mechanism is involved in regulating its localization. Our analysis suggests that the C-terminal tail of the kinase as well as its catalytic activity are responsible for the cytoplasmic localization. The relevance of this seems to be demonstrated by the fact that several RTT missense mutations within the catalytic domain generate kinase dead mutants or hypomorphic derivatives. Conversely, CDKL5 mutants carrying disease causing mutations truncating the protein in the C-terminus have increased catalytic activity and are costitutively mislocalized to the cell nucleus. Altogether these data suggest that not only loss of CDKL5 but also its gain of function is detrimental for nervous system functions and is involved in mental retardation/early seizure disorders including some cases of the RTT Hanefeld variant. Moreover, we believe that this analysis will contribute in drawing a phenotype-genotype correlation. Finally, in order to understand if MeCP2 is also an in vivo target of CDKL5 we performe in vitro phosphorylation assays and mass spectrometry to discover which residue(s) of the methyl-CpG-binding protein is phosphorylated by CDKL5.
Issue Date: 2009
Doctoral course: Biologia Cellulare e Molecolare
Academic cycle: 22
Publisher: Università degli Studi dell'Insubria
Citation: Giudici, L.The c-terminal tail and the catalytic activity of cdkl5 are fine regulators of its nucleo-cytoplasmic shuttling. (Doctoral Thesis, Università degli Studi dell'Insubria, 2009).

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