An allosteric switch between the activation loop and a c-terminal palindromic phosphomotif controls c-Src function.

Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phosphomotif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants.Weshow that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrateand enzyme-acting kinases during autophosphorylation.post-print5137 K

Determinantes estructurales y moleculares para la activacion y regulacion de c-Src mediante autofosforilacion

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 14-03-2024Phosphorylation is the chemical reaction by which a phosphate group is covalently bound to an organic molecule. This chemical modification is catalyzed by kinases, enzymes with phosphotransferase activity able to transfer the gamma-phosphate group of ATP onto the target molecule. Phosphorylation plays a fundamental role in the regulation of the functional and conformational states of enzymes. Kinases that phosphorylate other proteins as substrates (i.e. protein kinases) are crucial effectors in the regulation of most of the signalling pathways controlling almost every biological process within the cell. Intriguingly, many kinases are regulated by autophosphorylation, the process by which a protein phosphorylates to itself. Despite a widely described and accepted phenomena, the structural and molecular determinants that control protein autophosphorylation are not fully understood. In particular, the visualization and mechanistic dissection of the intramolecular versus the intermolecular components as well as the contribution of the catalytic versus the non-catalytic activities of protein kinases in the overall process. Furthermore, how autophosphorylation controls and regulates the transition between discrete conformational and functional states in proteins remains unclear. The perturbation of these processes by oncogenic mutations and other oncogenic insults are directly linked to cancer and other human diseases. Crucially, the structural and molecular understanding of this fundamental process can uncover vulnerabilities that can be therapeutically exploited for the design and development of more efficient and selective inhibitors of protein kinases. As a prototypical protein kinase and as a main paradigm for the work presented in this thesis we focus on the non-receptor tyrosine kinase c-Src. In the current paradigm for the regulation of c-Src tyrosine kinase activity, c-Src is held in an inhibited state by a phosphorylated Y530 at the c-terminal which engages the SH2 domain to adopt a close inactive conformation. This process is mediated by c-terminal Src kinase (CSK) and, upon phosphatase action (de-dephosphorylation) and in the presence of ATP, c-Src autophosphorylation on the activation loop promotes an active open state compatible with the phosphorylation of downstream substrates. In this thesis we show that contrary to the current paradigm, c-terminal Y530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Y419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Y530 autophosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Y530 intermolecular phosphorylation in which a c-terminal palindromic phospho-motif flanking Y530 on the substrate molecule engages the G-loop of the active kinase and adopts a position for ready entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src disfunction as indicated by viral and colorectal cancer (CRC) associated cterminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Y530 autophosphorylation and total phospho-tyrosine activity, and this detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a bi-directional crosstalk between the activation and c-terminal segments that controls the allosteric interplay between substrate and enzyme acting kinases during the process of autophosphorylationThe work of this thesis was carried out at the Spanish National Research Centre (CNIO) in the Kinases, Protein Phosphorylation and Cancer Group during the period from August 2018 to November 2022 under the supervision of Dr. Iván Plaza-Menacho, funded by an FPI-Severo Ochoa fellowship (BES-2017-082196) and grants (BFU2017- 86710-R, PID2020- 117580RB-I00 and RYC-2016-19382

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-ATPase (<i>PSMC</i>) Gene Family in the Light of Proteasome Inhibitor Resistance in Multiple Myeloma

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM