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Abstract
It has previously been found that the Akt pathway influences cholesterol
homeostasis through effects on the endoplasmic reticulum (ER) to Golgi transport of
the transcription factor, sterol-regulatory element binding protein-2 (SREBP-2).
SREBP-2 requires this transport for its activation. Here, we explored putative Akt
targets involved in this process.
A known Akt substrate was investigated, the mammalian target of rapamycin
(mTOR), and it did not affect SREBP-2 transport. However, it did affect the expression
of a downstream gene target of SREBP-2, the LDL-receptor, which may help explain
clinical observations of dyslipidaemia following treatment with clinical forms of
rapamycin.
A novel Akt substrate was identified, namely Sec24, an essential COPII
component involved in mediating cargo selection for ER to Golgi protein transport.
This was demonstrated clearly in two out of the four isoforms of Sec24 using a variety
of methods including in vitro and in vivo assays developed for this project. Many
techniques were also employed to help narrow down the particular phosphorylated
residue(s), including truncated and mutated plasmids and mass spectrometry. Further
work is required to investigate the remaining isoforms, as well as to determine the
phosphorylated residue(s). In addition, there appears to be functional consequences
of this phosphorylation, in that the binding of Sec23 to Sec24 is affected by the
phosphorylation status of Sec24.
Akt is an important signalling kinase involved in cell growth, survival, and
proliferation, and is often linked with cancer. Its phosphorylation of Sec24 may help
define Sec24's role in the regulation of protein transport through the early secretory
pathway, which is a relatively unexplored area.
Another novel Akt substrate was also uncovered, the RNA-dependent
RNA-polymerase (RdRp) from Norovirus, and the phosphorylation site has been
mapped. This is still a preliminary finding and further work is required to determine
the significance, which may include interesting effects on the virulence of the virus.