Live-cell Molecular Analysis of Akt Activation Reveals Roles for Activation Loop Phosphorylation

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization in response to shear flow can provide unprecedented opportunities for the molecular analysis of atherosclerosis. We carried out an extensive study of the design of molecular beacons to target BMP-4 mRNA, which plays important roles in proatherogenic development in response to unstable flow conditions. Specifically, we selected an optimal molecular beacon design, and found that the fluorescent intensity from targeting BMP-4 mRNA correlated well with the GFP signal after up-regulating BMP-4 and co-expressing GFP using adenovirus. The knock-down of BMP-4 mRNA using siRNA significantly reduced the beacon signal, further demonstrating detection specificity. We found that, due to target accessibility, molecular beacons designed with different target sequences gave very different signal levels, and establishing molecular beacon design rules has significant implications to live cell mRNA detections, especially to the studies of BMP-4 mRNA in endothelial cells under shear flow.

Download Full-text

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

Biochemical Journal ◽

10.1042/bj20150325 ◽

2015 ◽

Vol 471 (1) ◽

pp. 37-51 ◽

Cited By ~ 7

Author(s):

Tung O. Chan ◽

Jin Zhang ◽

Brian C. Tiegs ◽

Brian Blumhof ◽

Linda Yan ◽

...

Keyword(s):

Insulin Resistance ◽

Regulatory Mechanism ◽

Insulin Response ◽

Kinase Domain ◽

Activation Loop ◽

Akt Kinase ◽

Increase Insulin Sensitivity ◽

Akt Activation ◽

Kinase C ◽

Control Activation

This report identifies an allosteric regulatory mechanism involving C-terminal sequences and the Akt kinase domain that serves to restrict phosphatase access to the Akt activation loop. Importantly, this interaction can be modified to increase insulin sensitivity and protect cells from ceramide-mediated insulin resistance.

Download Full-text

Abstract 15889: C-Terminal Modifications of Akt Kinase Boost Cellular Responses to Insulin by Inhibiting Activation Loop Dephosphorylation

Circulation ◽

10.1161/circ.130.suppl_2.15889 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Tung Chan ◽

Jin Zhang ◽

Brian Tiegs ◽

Linda Yan ◽

Brian Blumhof ◽

...

Keyword(s):

Subcellular Location ◽

Cellular Responses ◽

Kinase Domain ◽

Intramolecular Interactions ◽

Activation Loop ◽

Cyclin Dependent Kinase ◽

Akt Kinase ◽

Akt Activation ◽

Hydrophobic Groove ◽

Insulin Dependent

Introduction: The Akt kinase isoforms (Akt1, Akt2 and Akt3) are activated downstream of the insulin receptor, and exert unique effects on cell growth, survival and metabolism. We recently described a novel ATP-dependent "dephosphorylation-resistance cage" in Akt kinases that controls access of cellular phosphatases to dephosphorylate the Akt activation loop (T308 in Akt1). Hypothesis: Here, we describe that, when Akt is localized at the cell membrane, intramolecular interactions of Akt C-terminal sequences with the hydrophobic groove of the kinase domain protect the phosphorylated activation loop (pT308 in Akt1) from cellular phosphatases. Methods and Results: Charged amino acid replacements of Akt C-terminal phosphorylation sites regulated by MTORC2 (Ser473) and by cyclin-dependent kinase 2 (Ser477) increased phospho-T308 protection from cellular phosphatases regardless of subcellular location. Functionally, these phosphatase-resistant Akt variants were refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent T308 phosphorylation in a regulated fashion (Figure 1). Conclusions: Collectively, these results suggest that modulating phosphatase sensitivity of Akt activation loops via C-terminal hydrophobic groove may provide a platform to develop novel Akt agonists.

Download Full-text