MIXED-LINEAGE KINASE 2-SH3 DOMAIN BINDS DYNAMIN AND GREATLY ENHANCES ACTIVATION OF GTPASE BY PHOSPHOLIPID

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expresse d 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 rol e of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S -transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding pro teins from rat brain extract. This analysis revealed that the major ML K2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By u sing two different forms of the dynamin proline-rich domain as affinit y 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 a ctivator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. W hen MLK2-SH3 was added to the assay together with PtdSer, however, dyn amin GTPase activity accelerated by more than 23-fold over basal level . An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved t ryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the sa me assay the SH3 domain from the regulatory subunit of phosphatidylino sitol 3'-kinase stimulated a similar synergistic acceleration of dynam in GTPase activity in the presence of PtdSer. These results suggest th at synergy between phospholipid and SH3 domain binding might be a gene ral mechanism for the regulation of GTP hydrolysis by dynamin.