Molecular Role of Dab2ip GTPase Activating Protein in Brain Development

Dab2ip is the member of Ras GTPase activating protein family that interacts with Disabled-1/2 famlily proteins, which play a critical role in brain development. Dab2ip is mainly known as a tumor suppressor, however recent studies have shown that it has important functions in the mammalian brain. In this study, using bioinformatics and experimental techniques, I characterized the mouse Dab2ip gene structure. Unlike previous reports, I found that the mouse Dab2ip gene has 20 exons and 3 CpG islands. I identified three transcript variants of Dab2ip which starts from exon one. Alternative splicing at the 5\u27 end of these transcripts result in the presence or absence of exon 3 and 5. Interestingly these two exons contain CpG islands 54 and 85, suggesting that methylation may control inclusion of these exons in the transcript. Quantitative RT-PCR and methylation experiments revealed that CpG 85 methylation increases during cerebellar development and positively correlates with inclusion of exon 5 in the Dab2ip transcript. Importantly, inclusion of exon 5 results in the expression of a full length PH domain in Dab2ip protein. In the second part of my studies, I examined the effect of the PH domain on Dab2ip GAP activity toward Ras and Rap1. I found that the presence of full length or the C-terminal half of the PH results in the specificity of Dab2ip toward Rap1 but not Ras. Live cell imaging showed that the Dab2ip PH domain allows it to activate Rap1 GTPase activity at both the plasma membrane and cytosolic structures. Thus, my results suggest that the presence of the PH domain may regulate Rap1 specificity, possibly by allowing Dab2ip to interact with membrane structures that are Rap1 enriched. Precise activation and inactivation of Rap1 signaling is critical for neuronal development. The increased DNA methylation and expression of Dab2ip transcript variant correspond to the period in cerebellar development when neurons are forming synapses. My results suggest that Rap1 signaling inactivation by Dab2ip plays an important role during this period of cerebellar development. Indeed my results are consistent with our previous observation that Dab2ip deficient animals have lower granule cell synapses