Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1.

Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.

TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development.

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Low expression of the putative tumour suppressor gene gravin in chronic myeloid leukaemia, myelodysplastic syndromes and acute myeloid leukaemia.

The putative tumour suppressor gene gravin is down-regulated in several solid tumours and is implicated in tumorigenesis. We have evaluated the expression levels of the gravin gene in the CD34(+)/blast cells of a range of myeloid malignancies as compared with controls using real-time quantitative polymerase chain reaction (PCR). Gravin was markedly down-regulated in 41 of 41 patients with acute myeloid leukaemia (AML), nine of 10 patients with myelodysplastic syndromes (MDS) and 33 of 33 patients with chronic myeloid leukaemia (CML), of whom 24 were in blast crisis (BC). We have shown that gravin is consistently down-regulated in the CD34(+)/blast cells of myeloid malignancies and may play a role in the molecular pathogenesis of these disorders.

Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology.

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal disorders of the haematopoietic stem cell and primarily involve cells of the myeloid lineage. Using cDNA microarrays comprising 6000 human genes, we studied the gene expression profiles in the neutrophils of 21 MDS patients, seven of which had the 5q- syndrome, and two acute myeloid leukaemia (AML) patients when compared with the neutrophils from pooled healthy controls. Data analysis showed a high level of heterogeneity of gene expression between MDS patients, most probably reflecting the underlying karyotypic and genetic heterogeneity. Nevertheless, several genes were commonly up or down-regulated in MDS. The most up-regulated genes included RAB20, ARG1, ZNF183 and ACPL. The RAB20 gene is a member of the Ras gene superfamily and ARG1 promotes cellular proliferation. The most down-regulated genes include COX2, CD18, FOS and IL7R. COX2 is anti-apoptotic and promotes cell survival. Many genes were identified that are differentially expressed in the different MDS subtypes and AML. A subset of genes was able to discriminate patients with the 5q- syndrome from patients with refractory anaemia and a normal karyotype. The microarray expression results for several genes were confirmed by real-time quantitative polymerase chain reaction. The MDS-specific expression changes identified are likely to be biologically important in the pathophysiology of this disorder.