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No previous studies have investigated the localisation of overexpressed human and mouse SGK1 isoforms. The findings in the present study showing immunostaining of overexpressed FLAG-tagged human and mouse SGK1 isoforms in transiently transfected HEK293T cells are summarised in Table 5.1. The majority of the human SGK1 isoforms and the equivalent mouse Sgk1 isoforms showed distinct similarity in their localisation. Human SGK1A and mouse Sgk1a displayed similar cytosolic network-like localisation. Human SGK1C and mouse Sgk1c both displayed similar diffuse localisation. Human SGK1D and mouse Sgk1d, both localised predominantly to the plasma membrane. Interestingly, human SGK1B and mouse Sgk1b were the only isoforms conserved between species that didn’t show similar localisation. Human SGK1B showed cytosolic network- like localisation similar to human SGK1A and mouse Sgk1a. In contrast, mouse Sgk1b displayed a predominantly nuclear localisation that was similar to human SGK1F, the only human isoform that is not conserved in mouse. These different localisations of SGK1 isoforms within species, yet similar localisations of equivalent SGK1 isoforms between species, suggest that they may have specific and conserved functional roles.

Although no previous studies have compared the localisation of all SGK1 isoforms before, several have investigated and found similar localisation of SGK1A alone. Naray-Fajes- Toth et al. (2004) showed that yellow fluorescent protein-tagged human SGK1A transfected into RCCT-28 cells (a rabbit CCD cell line) displayed a diffuse cytosolic localisation (Naray-Fejes-Toth et al., 2004a). Also, Menniti et al. (2005) showed that antibody detection of Myc-tagged human SGK1A transfected into COS7 cells also revealed a diffuse cytosolic localisation (Menniti et al., 2005). In a similar experiment,

(Arteaga et al., 2007). These three previous studies all used N-terminally tagged SGK1A constructs, but, in another study by Raikwar et al. (2008), comparable diffuse cytosolic localisation was shown of C-terminally FLAG-tagged human SGK1A in HEK293 cells (Raikwar et al., 2008). This study also showed that the regulated localisation of SGK1A to the cytosol is dependent on its N-terminus, as a 60 amino acid-truncated human SGK1A showed diffuse localisation throughout the cell (Raikwar et al., 2008). Raikwar et al (2008) also showed plasma membrane localisation of C-terminally FLAG-tagged human SGK1D in HEK293 cells (Raikwar et al., 2008). This shows that N-terminally and C- terminally FLAG-tagged human SGK1D localised identically in a similar cell type, suggesting that tag location did not affect localisation. The plasma membrane localisation of SGK1D may be due to its putative PX domain, which confers affinity for phosphoinositide binding. Arteaga et al. (2008) also described the localisation of mouse Sgk1d in transfected CHO cells as plasma membrane and showed that this was due to being bound to PI(4,5)P2 (Arteaga et al., 2008). This paper also found that removal of three positive residues in the mouse Sgk1d N-terminus by K21N/K22N/R23G mutations caused its translocation from the plasma membrane to the cytosol. Furthermore, F19A/F20A and W27A mutations caused Sgk1d translocation to the nucleus. This suggests that residues at positions 19-23 and 27 of Sgk1d, which are thought to be within the PX domain, are critical for its plasma membrane localisation. The plasma membrane localisation and/or phosphoinositide binding of SGK1D may confer its functional specificity.

Data from the present study showed that, although SGK1 isoform localisation in cultured cells can be similar to in native tissue (e.g. mouse Sgk1d), it can also be very different (e.g. human SGK1D). This may be because HEK293T cells are not representative of CCD cells, but could also be because cells were unpolarised, protein was overexpressed, or

isoforms were tagged. The attachment of any protein tag to the SGK1 isoforms may also interfere with their correct localisation. However, FLAG-tagging has the advantage that it involves the attachment of a relatively small epitope compared to fluorescent tags such a green fluorescent protein, whose larger size would increase the likelihood of localisation interference. A possible improvement to this investigation is to use polarised mpkCCDcl4 cells instead of HEK293T cells. Polarised mpkCCDcl4 cells would show the SGK1 isoform localisations that are specific to polarised CCD cells. Although both HEK293T cells and mpkCCDcl4 cells were used in this study, mpkCCDcl4 cells did not show high enough transfection efficiency for localisation experiments. HEK293T cells were kept in growth medium containing 10% FCS for SGK1 localisation studies, which should have stimulated SGK1 activity. Future studies into SGK1 isoforms to investigate effects on localisation could test the effects of treating serum-starved HEK and CCD cells with aldosterone on localisation of FLAG-tagged SGK1 isoforms. Investigation of the changes in SGK1 isoform localisation in response to their phosphorylation and activation may reveal more about the changes regarding which isoforms are involved in regulation of ENaC. However, this approach still relies on transient transfection, which floods the cell with overexpressed protein possibly causing altered or disregulated localisation. Future experiments could target untagged, overexpressed, or endogenous protein, but this requires detection using isoform-specific antibodies. In a previous study, Sahoo et al. (2005) showed that endogenous SGK1 localisation in human breast cancer cells was predominantly cytosolic and network-like, using a pan-SGK1 antibody, suggesting that this staining was accounted for by SGK1A and SGK1B (Sahoo et al., 2005). The human SGK1 isoform-specific antibodies described in this thesis showed decreased specificity when used to detect untagged protein for immunofluorescence. Generation of high-avidity

SGK1 isoform-specific antibodies would allow future investigation of differential isoform regulation and localisation using immunofluorescence.