PROTEIN INTERACTIONS AND FUNCTION OF IQ MOTIF-CONTAINING PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A number of unidentified IQ motif-containing proteins of unknown biological function occur in cells. Three of these putative calmodulin-binding proteins are ASPM (abnormal spindle primary microcephaly), KIAA1023 and KIAA0036. These proteins have multiple IQ motifs, are widely expressed in cells and appear important to mitosis and development. ASPM has recently been found to be associated with microcephaly in humans and appears to interact with the mitotic spindle based on its orthology to the mouse SHA1 protein and Drosophila ASP which also contain multiple IQ motifs. ASPM is one of six loci associated with primary microcephaly in humans and appears to be involved in developmental neurogenesis. In 2005, the second gene KIAA0036 was linked genetically to cystic kidney disease or nephronophthisis-5 that leads to chronic renal failure and retinitis pigmentosum in the young. Work completed on this project involves measurement of mRNA expression of the three multiple IQ-motif genes in different human cell lines and in adult tissues. These genes occur in nearly all transformed human cell lines examined. All three genes were highly expressed in over sixteen human tissues with the exception of adult brain and skeletal muscle where only ASPM levels were negligible. Studies by others failed to find expression of the ASPM gene in different regions of adult mouse brain such as olfactory bulb, hippocampus and cortex. Some of these regions in the adult are known to undergo neurogenesis. We earlier identified the ASPM gene in a number of primates. Comparisons with the mouse sequence in two selected regions of exon 18 indicated that ASPM in primates did not exhibit any of the deletions that occur in rodents. In the specific regions sequenced, the IQ motifs were similar in number, placement and sequence suggesting that all primates have very analogous proteins. This suggests that differences in the sequence of the ASPM gene, particularly in the IQ region are not responsible for the increase in the size of the cortex among primates as suggested by some laboratories. This data is now supported by the complete sequencing of the ASPM gene in a number of primates by other laboratories. A pre-doctoral candidate in this laboratory is also examining the role of the ASPM gene in adult and developing zebrafish in collaboration with the University of Oregon. It is hypothesized that ASPM is important in the development of the brain in zebrafish. Morpholino anti-sense oligonucleotides were used to knockdown the gene during embryogenesis and investigate the effects on morphology of the brain and marker enzymes. Additionally, a number of specific primers were developed for sequencing missing regions of the zebrafish gene and for the preparation of RNA probes for in situ hybridization. We have also conducted bioinformatic studies to characterize the putative microtubule binding region of ASPM and to examine the multidomain structure of ASPM family members. With regard to species distribution, ASPM appears to be present in all vertebrate and in some invertebrates and plants, but is absent in yeast. Efforts are also focused on expressing in bacteria the different regions of these proteins (i.e., IQ motifs, SHA-specific region, and actin-binding regions) to examine potential binding partners and functionality of these regions. Although two regions of ASPM have been cloned into the pet15b vector, expression of the protein has not been demonstrated. Cloning and other techniques such as the two-hybrid system are being examined in attempts to define protein interactions and the function of these genes. Interaction studies are critical to understanding the function of these proteins in mitosis and development.