Characterization Of EEF1Bγ And SRm160 As Substrates Of The LAMMER Protein Kinase DOA Of Drosophila

The Darkener of apricot(Doa) is the unique locus encoding LAMMER kinase in Drosophila. LAMMER kinase is also known as CLK for its 4 paralogues(CLK1-4) in mammals.The best studied role of LAMMER kinases is that it phosphorylate the SR and SR-like proteins and subsequently regulate the alternative splicing events in somatic sex-determination pathway in Drosophila.In this study,we identified eEF1Bγand SRm160 as the substrates of DOA kinase,and characterize their roles in development of Drosophila,in order to shed more light on the roles of LAMMER kinase.Drosophila eEF1Bγwas isolated as a protein interacting with DOA kinase in a yeast two-hybrid screen.It contains a predicted and conserved phosphorylation site(Serine²⁹⁴) for LAMMER kinases in its C-terminal region.The locus encoding eEF1Bγin Drosophila or any other higher eukaryote has never been analyzed,eEF1Bγprotein is phosphorylated in vitro and as well in vivo by DOA,and the phosphorylation site identified via site-directed mutagensis.The protein expression pattern in embryos is investigated via immunocytochemistry and the existence of EF-1γis detected in pupal nuclear fractions.I generated and characterized several EF-1γmutant alleles via excision of a P-element provides,including a recessive lethal null allele.To define further the function of the phosphorylation of EF-1γby DOA,we generated wildtype and mutant constructs expressing a Serine-294-Alanine substitution.The absence of this phosphorylation site reduces the activity of the protein,since the expression of the mutant construct does not rescue the lethality caused by homozygoty of the null allele as well as that of the wildtype.SRm160 functions as a splicing co-activator,which is required in both constitutive and alternative splicing.It is also involved in pre-mRNA 3'-end processing.SRm160 contains several potential phosphorylation sites for LAMMER kinases.The transcript and protein expression pattern of SRm160 during embryogenesis were performed.Loss of SRm160 activity and its ubiquitous over-expression provoke lethality.Not surprisingly,over-expression and RNA interference for SRm160 with different drivers disturbs the development of multiple organs.RNA interference of SRm160 leads to slightly rough eyes and blistered wings,respectively.Overexpression SRm160 result in extremely rough eyes and abnormal genital structure or males lacking genitalia and analia, respectively.Interestingly,Doa mutations supress the overexpression phenotype in eye,but enhance the phenotype in genitals,indicating different singal pathways are involved in the interaction.In addition,as for the interaction between Doa and SRm160,SRm160 mutant alleles enhance the female-to-male sex transformation phenotype of Doa alleles,further indicating its involvement in somatic sex determination,and its probability as a DOA substrate.Our results to date indicate the strong genetic interactions between Doa and SRm160,suggesting the idea that SRm160 is a substrate of DOA kinase.However,more biochemical and molecular investigations need to be done to elucidate and confirm the mechanism of their interactions.