A number of unidentified IQ motif-containing proteins of unknown biological function occur in cells. Two of these putative Ca2+-independent calmodulin (CaM)-binding proteins in humans are FLJ10517 (FLJ-IQ) and KIAA0036 (Kazusa cDNA project).
Additional unidentified human IQ motif-containing proteins also occur in the database. Both proteins have multiple IQ motifs and are widely expressed in cells. FLJ-IQ, the putative orthologue of mouse SHA1 exhibits 70-80 % identity to the last 200 residues of the C-terminus of the protein and also contains multiple IQ motifs. SHA1 is found associated with the polar region of the mitotic spindle implicating a role for CaM in controlling mitotic microtubular activity.
The other protein, KIAA0036, was cloned from an immature myeloid cell line and contains a pore-like region that is characteristic of a Trp or capacitative calcium entry channel, but lacking the characteristic six transmembrane domains of the Trp channel.
To elucidate the function of these proteins, expression of the mRNA of the human/mouse orthologues in different tissues and cells is examined by Northern blot analysis to determine the prevalence and size of the transcript. Expression of mRNAs will be examined in response to Jurkat cell activation by thapsigargin (TG) or by diacylglycerol, Ca2+-specific ionophore treatment. Gene constructs (cloned from a human cDNA library) with an in-frame fusion of a sequence coding for the influenza hemaglutinin epitope will be utilized for transfection.
The expressed proteins together with extracts of potential binding partner protein(s) including CaM will be immunoprecipitated, fractionated, and the partners identified by mass spectroscopy or by Western blot in the case of CaM. A search for binding partners may also be undertaken using the yeast 2-hybrid system.
Finally, cellular localization of the proteins in response to cell activation or treatment with TG, diacylglycerol or ionomycin is examined using a green fluorescent protein fusion of each protein.
The RCMI facilities of the core laboratories of Molecular Computations and Bioinformatics are utilized in these studies to further examine the family of IQ proteins for verification of cloned genes and PCR products. These molecular studies should decipher the function of these widely expressed and putative CaM-regulated proteins. This project utilizes resources of the LMCB.