Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster.
Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. We found that at least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, we undertook a characterization of the system. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function.
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Biochemistry Graduate Program, Department of Biology and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Griffith
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D004331:Drosophila melanogaster / Q000378:metabolism*; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D025842:SUMO-1 Protein; D044764:Ubiquitin-Activating Enzymes; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D011506:Proteins; D025842:SUMO-1 Protein; C470301:UBA2 protein, human; D014452:Ubiquitins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D044764:Ubiquitin-Activating Enzymes