Dept of biology and center of complex system, Brandeis University,
415 South St.,
Waltham, MA, 02154, U.S.A.
Ca2 /calmodulin-dependent kinase II has been suggested to be a multifunctional enzyme and a variety of molecules have been shown to be substrates in vitro. In mammals and Drosophila this enzyme is mainly localized in the brain and plays a central role in the regulation of neuronal plasticity. To investigate the in vivo function of CaM kinase II in Drosophila, we used a genetic approach to isolate upstream and/or downstream components of the CaM kinase II pathway. We searched for second site mutations that act as enhancers of lethality in CaMKII heterozygotes. This mutant was crossed with recessive lethal mutants generated by insertion of a P-element (Berkley and Kiss collections). Enhancers are defined as those strains for which the transheterozygote flies generated by the cross are less than 50% less viable compared to the rest of the progeny. From this screen, we isolated two strains (#4065 and #4530) which are strong enhancers: the % of viability of the transheterozygotes are similar and low (around of 16%). In the 4065 strain, the transposon is inserted 680bp from the ATG of the gene encoding for transcription factor Adf1 (England et al., 1992, Proc.Natl.Acad.Sci., 89, 683-687). We tested if Adf1 could be a substrate of the CaM kinase II in a phosphorylation assay. Adf1 is indeed phosphorylated by the CaM kinase II and chymotrypsin treatment of Adf1 shows that the phosphorylation occurs in the N-terminal which contains the DNA binding domain. In order to show that the 4065 strain is a Adf1 mutant, we are now trying to rescue the lethality of this mutant with a wild type copy of the Adf1 gene. The two strains #4065 and 4530 have been shown to be identical for several different phenotypes: 1) Lethal stage is between the 1st and 2nd larval instar, 2) b-galactosidase staining is seen in the CNS of embryos and larvae and in adult heads is localized around the lamina and the lobula, and around the CNS 3) The viable transhetrozygotes from the cross between CaMKII and each mutant exhibit alterations in wing shape, and finally 4) The two mutations do not complement each other (0/143). However, the two insertions do not map to the same region according to the Berkeley project. #4065 maps at the 41C1-2 on polytene chromosome while the #4530 is at 38F5-6. These results suggest that either the mapping is erroneous or the Adf1 gene has a spread out structure in the genome or the insertion in the 4530 strain affects a gene whose product interacts strongly with Adf1 in a pathway involving CaM kinase II. Behavioral tests and physiological studies at the larval muscular junction will be done on these strains to further investigate their interation with CaM kinase II.