Functional Characterization Of A Cardiac-specific Kinase P93 In Cardiac Development And Structural Heart Diseases

It is estimated that a proportion of the 20,900-27,160 genes comprise cardiovascular genome(C C Liew, personal communication), and cardiovascular disease-associated genes which derive from them play pivotal roles in cardiovascular system. Cloning and characterization of these genes may help to understand the mechanisms of cardiovascular developmental and pathophysiological processes, which can serve as the basis for clinical treatment for cardiovascular diseases.Efforts in the human genome project over the last decade have created a stream of opportunities for a fundamentally collaborative discipline bioinformatics/computational biology. Combined with the novel ESTs(expressed sequence tags) isolation on the basis of constructed human heart cDNA library, bioinformatics was employed to identify cardiovascular-associated novel genes. Here, two full-length cDNAs, i.e., cardiac-specific kinase p93 and muscle-specific NELIN, are described in brief.A novel kinase gene localized on 1p31.1 within AVSD (atrioventricular septal defect) critical region is identified. The 3420bp clone contains one 2,535bp open reading frame, which encodes 835 amino acids product with a predicted Mr of 93 kDa and we termed it p93. p93 contains three kinds of protein domains: 7 N-terminal ankyrin repeats, a protein kinase domain; and a C-terminal Ser-rich domain. Homology search suggested that p93 is a distant family member of ILK(integrin-linked kinase). Further inspection of the sequence by phylogenetic analysis indicated that p93 and ILK may belong to MAPKKKs superfamily.A single 3.42-kb transcript was readily detected in heart tissues by multiple fetal and adult tissue Northern Blot. Subsequently 76-tissue array analysis showed that p93 is expressed in fetal and adult heart, and its expression profile in adult heart as follows: strongly expressed in interventricular septum and apex of the heart in comparison with right atrioventricular part, and even less strongly in left ventricle, weakly in left atrium, and undetectably in any other tissues, which suggested p93 is virtually cardiac-specific.Polyclonal antibodies were produced in rabbit through gene-based vaccines, and its titer and specificity were assessed by prokaryotic expression of p93 protein. Heart immunohistochemistry revealed that p93 is predominantly expressed in the nucleus of embryonic and adult cardiac myoctyes, while it is undetectable in smooth muscle cells and endothelial cells of vasculature.