Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid

被引:23
作者
Rasmussen, RK
Rusak, J
Price, G
Robinson, PJ
Simpson, RJ
Dorow, DS
机构
[1] Peter MacCallum Canc Inst, Trescowthick Res Ctr, Melbourne, Vic 3000, Australia
[2] PO Royal Melbourne Hosp, Walter & Eliza Hall Inst Med Res, Parkville, Vic 3052, Australia
[3] PO Royal Melbourne Hosp, Ludwig Inst Canc Res, Joint Prot Struct Lab, Parkville, Vic 3052, Australia
[4] Childrens Med Res Inst, Cell Signalling Unit, Sydney, NSW 2145, Australia
关键词
D O I
10.1042/bj3350119
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
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页码:119 / 124
页数:6
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