Abstract
The serine-threonine kinase AKT/PKB is a critical regulator of various essential cellular processes, and dysregulation of AKT has been implicated in many diseases, including cancer. Despite AKT action is known to function mainly in the cytoplasm, AKT has been reported to translocate to the nucleus. However, very little is known about the mechanism required for the nuclear import of AKT as well as its function in this cellular compartment. In the present study, we characterized the presence of endogenous nuclear AKT in human melanoma cells and addressed the possible role of AKT by exploring its potential association with key interaction nuclear partners. Confocal and Western blot analyses showed that both phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei. Using mass spectrometry in combination with protein-crosslinking and co-immunoprecipitation, we identified a series of putative protein partners of nuclear AKT, including heterogeneous nuclear ribonucleoprotein (hnRNP), cytoskeleton proteins β-actin, γ-actin, β-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated β-actin as a new nuclear AKT-interacting partner. Cofilin and active RNA Polymerase II, two proteins that have been described to interact and work in concert with nuclear actin in transcription regulation, were also found associated with nuclear AKT. Overall, the present study uncovered a yet unrecognized nuclear coupling of AKT and provides insights into the involvement of AKT in the interaction network of nuclear actin.
Highlights
The serine-threonine kinase AKT is a critical regulator of various essential physiological cellular processes including cell proliferation, survival, motility, metabolism and differentiation [1]
To confirm that phosphorylation of AKT is not required for interaction with β-actin, we examined whether recruitment of β-actin by nuclear AKT was sensitive to phosphoinositide 3-kinase (PI3K) inhibition
By co-IP analysis we demonstrated that AKT/β-actin coupling was not disturbed by inhibition of AKT phosphorylation, since β-actin was still interacting with AKT in the presence of the PI3K inhibitor XI, suggesting that nuclear AKT/β-actin complex is formed regardless of AKT-Ser473 phosphorylation state (Figure 5D)
Summary
The serine-threonine kinase AKT ( named protein kinase B, PKB) is a critical regulator of various essential physiological cellular processes including cell proliferation, survival, motility, metabolism and differentiation [1]. It is well established that a multi-step process activates AKT, comprising a cascade of events that depend on PtdIns-3-4-5-P3 (PIP3), which are products of phosphoinositide 3-kinase (PI3K). Interaction of PIP3 with the pleckstrin homology (PH) domain of AKT brings AKT close to the plasma membrane and leads to a conformational change, which converts AKT as a substrate for regulatory kinases such as phosphoinositide-dependent kinase 1 (PDK-1) that phosphorylates AKT at Thr308. AKT translocates from the inner surface of the cell membrane to other cell compartments to interact and phosphorylate a wide spectrum of substrates that regulate a diversity of cellular process [5]
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