2016Joint Usage and Research Report
Title of Research Project
ApplicantRegulation of Akt by a novel ubiquitin E3 Ligase, RNF144A

Institution

King’s College London

Job title

Principal Investigator/Sr. Lecturer

and name
Visiting

NameDr. Susan John

researcher
Purpose of the Research Project

AKT (or protein kinase B, PKB), is a serine/threonine protein(approx. 250 words)

kinase that is activated downstream of the lipid kinase
Phosphoinositide 3-kinase (PI3K) following growth factor,
cytokine or antigen receptor stimulation. AKT is a criticalsignalling molecule that regulates and coordinates diverse
cellular functions such as, cell size, proliferation, survival,translation, metabolism, and motility. The critical function ofAKT in maintaining normal cellular homeostasis isunderscored by human diseases such as cancers, metabolic
disorders, Down’s Syndrome and Type-1 Diabetes, where
AKT function is either hyperactivated or inactivated. In lightof its vital role in controlling cellular homeostasis, AKT
function is tightly controlled under normal physiologicalconditions. One important regulatory mechanism by which
AKT activity is regulated is ubiquitylation. To date a few
different E3-Ubiquitin ligases have been identified which canubiquitylate AKT or the active phosphorylated-AKT resulting
in either proteasomal degradation or promote nuclearlocalization. We have recently, identified a novel cytokine
induced E3-Ub ligase, RNF144A, whose activity inhibits theAKT/mTOR pathway. In this collaborative study we wish toevaluate if there is a physical and functional interaction
between AKT and RNF144A and whether RNF144A
regulates Akt mediated autophagy in lysosomes.

Development of the Research

The physical interaction between AKT (plasmid kindly

Project and Results

provided by Prof.Noguchi) and RNF144A was investigated.

(approx.. 850 words)

293T

cells

weretransfected

withFLAG-AKTandMyc-RNF144A

andimmunoprecipitatedtotalwithlysates
aprepared

FLAG

antibody

andtoimmunoprecipitate AKT. The immunoprecipitates weresubjected to SDS-PAGE and western blot analysis performed
with an anti-myc antibody to detect any co-precipitating
RNF144A. The results showed specific co-precipitation ofRNF144A with AKT, suggesting that the two proteins caninteract together in a complex.
Given that there is a physical interaction between the twoproteins, we next investigated whether RNF144A canubiquitinateAKT.

293Tcells

weretransfected,

with

myc-RNF144A and FLAG-AKT, or either plasmid alone or
empty vector alone. Immunoprecipitation analysis wasperformed using an anti- FLAG antibody to precipitate AKT,
followed by SDS-PAGE and western blot analysis to detectubiquitination using an anti-ubiquitin antibody (VU-1).
The ubiquitin antibody detected polyubiquitination of AKT
when RNF144A was co-expressed with it, but not in either
control samples. Furthermore, the addition of a proteasomal
inhibitor MG132 to the transfected cells showed increased
AKTproteinlevels

whenRNF144A andAKT wereco-expressed. This result suggested that RNF144A could
induce polyubiquitination leading to proteasomal degradationof AKT. Thus we have made the novel observation thatRNF144A is an E3-Ubiquitin ligase that potentially regulates
AKT levels. We will next, confirm the nature of the ubiquitin
linkage on AKT by using lysine mutant constructs ofubiquitin.
Based on these results, experiments have been planned
during Dr. John’s visit to Prof. Noguchi’s laboratory at
Hokkaido University to investigate the functional impact of
the regulation of AKT by RNF144A in autophagy. Prof.
Noguchi’s laboratory has recently identified a role for AKT in
autophagy mediated by lysosomes. RNF144A knockout cells
show a gene expression profile that is indicative ofdysregulation of the autophagic process and increased AKT
levels, and therefore we hypothesize that the RNF144A
mediated degradation of AKT will be important for AKT
function in lysosomes. In the future work planned, Prof.
Noguchi’s laboratory will assess the co-localization of

RNF144A and AKT in lysosomes, and the function of AKT inlysosme mediated autophagy in RNF144A knockout cells
using lentiviral mediated delivery of shRNA to knockdown
RNF144A (lentiviral vector provided by Dr. S.John).
Thus, our studies currently identify AKT to be a novel
substrate for RNF144A with potential important functional
implications for cellular processes regulated by AKT, such asautophagy and metabolism.

Publication【Conference, symposium, workshop etc.】*Enter the names of conferenceor journal and its vol. No. whereWork still in progress. Too early to publish yet.

the above work was presented.

【Journals】