Identification and characterisation of a novel variant of serum and glucocorticoid regulated kinase-1 with an N-terminal phox homology-like domain

• Main Author: Dunmore, R.
• Published: University of Cambridge 2008
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598689

Serum and glucocorticoid regulated kinase 1 (SGK1) is a serine/threonine kinase whose function is controlled at the levels of transcription, subcellular localisation and phosphorylation-dependent activation in a tissue-specific and stimulus-dependent manner. Existence of a second form of SKG1 (in this thesis termed SGK1b) was suggested by identification of an mRNA (AK055088) coding for a putative kinase differing from SGK1 only in the N-terminal region. Bioinformatic analysis revealed that this mRNA is the result of transcription from an alternative initiation site where splicing produces an mRNA in which exon 1 of SGK1 is replaced with 3 exons from a coding region proximal to this site.  Taqman analysis showed SGK1b mRNA was widely expressed in all human tissues tested. At the protein level, the N-terminal region of SGK1b was predicted (from primary sequence) to form a phox homology (PX) domain-like structure. This domain was expressed in bacteria. Lipid overlay assays demonstrated specific phosphoinositide binding which, surprisingly, was not disrupted by alteration of conserved arginines at position 127 and 127. Subcellular localisation studies on protein expressed in mammalian cells showed both isoforms of SGK1 were present throughout the cell, although SGK1b uniquely showed some nuclear and plasma membrane localisation. SGK1b kinase activity, which was sensitive to an SGK1 inhibitor, was detected in HeLa cells overexpressing wild type or constitutively active, but not kinase-dead SGK1b. Western blotting, with a range of SGK1b specific antibodies were unable to detect endogenous expression of SGK1b possible due to low level expression. These studies thus identify SGK1b as a novel protein kinase with a unique N-terminal region which demonstrates PX domain-like properties. SGK1b also shows different patterns of expression and subcellular localisation from those of SGK1 suggesting this protein may be regulated in a different manner.