Falke, J. J. | Search bioRxiv

bioRxiv2021

HPLC Method to Resolve, Identify and Quantify Guanine Nucleotides Bound to the GTPase Ras

The Ras superfamily of small G proteins play central roles in diverse signaling pathways. Superfamily members act as molecular on-off switches defined by their occupancy with GTP or GDP, respectively. In vitro functional studies require loading with a hydrolysis-resistant GTP analogue to increase the on-state lifetime, as well as knowledge of fractional loading with activating and inactivating nucleotides. The present study describes a method combining elements of previous approaches with new, optimized features to analyze the bound nucleotide composition of a G protein loaded with activating (GMPPNP) or inactivating (GDP) nucleotide. After nucleotide loading, the complex is washed to remove unbound nucleotides then bound nucleotides are heat-extracted and subjected to ion-paired, reverse-phase HPLC-UV to resolve, identify and quantify the individual nucleotide components. These data enable back-calculation to the nucleotide composition and fractional activation of the original, washed G protein population prior to heat extraction. The method is highly reproducible. Application to multiple HRas preparations and mutants confirms its ability to fully extract and analyze bound nucleotides, and to resolve the fractional on- and off-state populations. Furthermore, the findings yield a novel hypothesis for the molecular disease mechanism of Ras mutations at the E63 and Y64 positions.

cancer biology↗

bioRxiv2021

Single Molecule Studies Reveal Regulatory Interactions between Master Kinases PDK1, AKT1 and PKCalpha

Leukocyte migration is controlled by a leading edge chemosensory pathway that generates the regulatory lipid PIP3, a growth signal, thereby driving leading edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1/PKB and PKC are key components of a plasma membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, while PKC is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKC and other AGC kinases by phosphorylation at key sites. PDK1 is known to form PDK1:AKT1 and PDK1:PKC heterodimers stabilized by a PIF interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here we present the first, to our knowledge, single molecule studies of full length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full length PKC. The findings show that membrane-bound PDK1:AKT1 and PDK1:PKC heterodimers form under physiological conditions, and are stabilized by PIF interaction. PKC exhibits 8-fold higher PDK1 affinity than AKT1, thus PKC competitively displaces AKT1 from PDK1:AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1:AKT1 heterodimer, while PKC acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide new insights into molecular and regulatory interactions of the three master kinases on their target membrane, and suggest that the recently described tumor suppressor activity of PKC may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis. STATEMENT OF SIGNIFICANCEThis work investigates three master kinases that play central roles in guiding white blood cell migration to sites of infection, inflammation or tissue damage. More broadly, the same kinases help regulate production of a cell growth signal, and may trigger cancer when dysregulated. Using powerful single molecule methods, the work detects and analyzes the interactions between the three purified kinases on their target membrane surface. The findings reveal functionally important differences between pairwise binding affinities of different binding partners. Additional studies reveal that the highest affinity kinase can disrupt and inhibit the activated complex formed by association of the other two kinases. Such inhibition is proposed to help prevent cancer by limiting growth signal production by the activated complex.

biophysics↗