Through endocrine and exocrine functioning, physiological needs are
communicated to body systems. Physiological need is met through the actions of
intracellular signaling cascades and calibrated through an extensive network of regulatory
cross talk within the cells of a given tissue.
The insulin receptor belongs to a family of perhaps one of the most well studied
family of dual receptor and tyrosine kinases (RTK). The signaling cascade downstream
of the insulin RTK can be initiated through Insulin or growth factor ligand binding and
bears growing relevance to the projected epidemic of obesity related illness and
associated cancers. The primary function of the post-prandial insulin response is to
support nutrient uptake and storage.
Insulin (IS), Insulin-Like Growth Factor (IGF), and Epidermal Growth Factors
(EGF) contribute to glucose metabolism, energetic homeostasis, and anabolic
applications through effector kinases downstream of activated (phosphorylated) insulin
receptor substrates (IRS). Protein Kinase B (AKT) kinase is one such cytosolic effector
known to be of critical importance to anabolic metabolism and general cell survival.
Under normal circumstances, AKT activity is dependent upon dual phosphorylation
events known to occur at the plasma membrane.
In an attempt to better understand the mechanism of AKT recruitment to the plasma membrane, earlier experiments reported that IRS stimulation by Insulin-Like Growth Factors (IGF) and Epidermal Growth Factors (EGF) resulted in downstream poly-ubiquitination and subsequent activation of the AKT kinase. This sequence of post-translational modification events suggested that non-proteolytic AKT ubiquitination, accomplished by the E2 Ubiquitin Conjugating enzyme (UBC13), was an important mediator of AKT activation.
Through subsequent experimentation, it was determined that non-proteolytic ubiquitination was a necessary step for AKT activation following IRS activation by Insulin. Furthermore, the same two sites previously described in the context of IGF/EGF signaling were exploited through targeted mutagenesis and shown to synergistically regulate AKT translocation to the plasma membrane. Mutant AKT variants with a single mutation to either ubiquitination site resulted in partial knock down of phosphorylated AKT (pAKT), while variants with double mutations resulted in a complete loss of pAKT detection.
Under physiologic conditions UBC13 activity can be antagonized by a small multifunctional protein called G-Protein Pathway Suppressor 2 (GPS2). Bearing the kinetics of an endogenous inhibitor, GPS2-mediated regulation directly inhibits the ubiquitin conjugating activity of the enzyme; thereby restricting AKT non-proteolytic poly-ubiquitination and antagonizing the insulin signaling network through a conserved mechanism. In accordance with this role, we have previously shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance.
As we are just beginning to unravel the regulatory network governing the cellular response to nutrient excess and pro-growth signaling, it remains unclear whether UBC13 activity is universally engaged in AKT translocation and activation. Here we have focused on the mitochondrial pool of AKT and investigated its regulation. Our findings add to the growing body of knowledge by demonstrating that in pre-adipocytes mitochondrial AKT is activated, in a UBC13-dependent fashion, following insulin stimulation. We also show that GPS2-mediated inhibition of UBC13 equally antagonizes AKT activation in different subcellular compartments, and that mitochondrial AKT activation is partially Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) dependent.
Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/37014 |
Date | 19 June 2019 |
Creators | Shambley, Aaron |
Contributors | Perissi, Valentina |
Source Sets | Boston University |
Language | en_US |
Detected Language | English |
Type | Thesis/Dissertation |
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