Author Yong, Thomas Joon Kin
Title Molecular and biochemical characterisation of human Casein Kinase I Alpha splice variants.
Institute Thesis (Ph.D.) National Institute of Education, Nanyang Technological University
Year 1998
Supervisor Gan, Yik Yuen
Call no. QP606.P76 Yon
 
Summary
Casein Kinase I (CKI), one of  the first protein kinases identified biochemically, is a serine/threonine protein kinase known to exist in multiple isoforms in mammals. It has been shown in mammalian tissues that CKIalpha exists in two or three alternatively spliced variants (Rowles et al., 1991; Zhang et al., 1996; kuret et al., 1997). Recently, four alternatively spliced variants of CKI alpha (CKla, CKlas,CKla LS, CKlaL) were identified in diverse chicken cell types (Green and Bennett, 1998). To date, only a human CKlaS cDNA has been published (tapia et al., 1994; fish et al 1995). Here, I report on the isolation of near full length cDNAs coding for human CKlaS and human CKlaL derived from the 4.2 kb mRNA transcripts. Both splice variants are also likely to be encoded by the 2.4 kb mRNA transcripts, generated by employing an alternate polyadenylation signal identified in the longer transcripts. The 4.2 kb mRNA species of both splice variants contain   five RNA-destabilising AU-rich element (ARE) motifs in their  3' untranslated regions, the 2.4 kb transcripts contrain a single ARE; suggesting the longer transcripts may be more rapidly turned over. Compared human CKlaS, human CKlaL contains an additional 28 amino acids after Lys152, and a deletion of 12 amino acids after Gly323. In addition, a cDNA coding a potential novel splice variant of human CKlaL, which I have named human CKlaT, has been identified . Human CKlaT lacks the catalytic subdomains I to Vlb ( as defined by Hanks st al., 1988) but contains a nine novel amino acids at the amino terminal, followed by the 28 amino acids identified as the 'L' insertion and the subdomains VII to XI of the catalytic domain. The regulatory domain of human CKlaT is identical to that of human CKlaS but contains an additional  eight amino acids at the carboxyl terminal not previously reported in any CKlalpha. Evidence from attempts at isolating additional independent CKlaT to the human regulator of mitotic spindle assembly-1 (RMSA-1), Northern analysis, and immunoblotting experiments, suggest that CKlaT cDNA may be a cloning artefact. Human CKlaS and CKlaL splice variants fused to the maIE bacterial protein have knase activity. CKlaL had a preference for phosvitin over casein as a substrate but a higher kinetic constant (Km) for casein than for phosvitin, while the reverse was observed for CKlas. Expression of human CKlat splice variant fused to the bacterial maIE protein demonstrated that CKlat had limited kinase activity for casein and no activity for phosvitin. Using  antibody specific to the CKI alpha L variants, I found subcellular distribution of the CKI alpha L variants is cell cycle dependent in human  HEp2 cells. At interphase, CKI alpha L variants  are restricted to sytisolic membranes , with no staining of the centrosome or nucleus. In mitotic cells, staining of the cytosolic membranes is lost and cki alpha L variants localise intensely at the mitotic centromsomes , with additional staining to the microtubules.