Paxillin phosphorylation at serine 273 and its effects on Rac, Rho and adhesion dynamics

Abstract

Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are spatiotemporally regulated, with new adhesions forming at the leading edge of the cell and mature adhesions disassembling at the rear. Signalling proteins and structural cytoskeletal components tightly regulate adhesion dynamics. Paxillin, an adaptor protein within adhesions, is one of these proteins. Its phosphorylation at serine 273 (S273) is crucial for maintaining fast adhesion assembly and disassembly. Paxillin is known to bind to a GIT1-βPIX-PAK1 complex, which increases the local activation of the small GTPase Rac. To understand quantitatively the behaviour of this system and how it relates to adhesion assembly/disassembly, we developed a mathematical model describing the dynamics of the small GTPases Rac and Rho as determined by paxillin S273 phosphorylation. Our model revealed that the system possesses bistability, where switching between uninduced (active Rho) and induced (active Rac) states can occur through a change in rate of paxillin phosphorylation or PAK1 activation. The bistable switch is characterized by the presence of memory, minimal change in the levels of active Rac and Rho within the induced and uninduced states, respectively, and the limited regime of monostability associated with the uninduced state. These results were validated experimentally by showing the presence of bimodality in adhesion assembly and disassembly rates, and demonstrating that Rac activity increases after treating Chinese Hamster Ovary cells with okadaic acid (a paxillin phosphatase inhibitor), followed by a modest recovery after 20 min washout. Spatial gradients of phosphorylated paxillin in a reaction-diffusion model gave rise to distinct regions of Rac and Rho activities, resembling polarization of a cell into front and rear. Perturbing several parameters of the model also revealed important insights into how signalling components upstream and downstream of paxillin phosphorylation affect dynamics.

Paxillin S273 Phosphorylation Regulates Adhesion Dynamics and Cell Migration through a Common Protein Complex with PAK1 and βPIX

“…In this study, we identified two paxillin-WT adhesion subpopulations experimentally: one was small dynamic adhesions and one was larger stable adhesions 48 . This bimodality was abolished experimentally with expression of paxillin-S273D or S273A mutants, which had predominantly small, dynamic or large, stable adhesions, respectively 48 . The model also identified a bistable switch consistent with the two populations of adhesions, dynamic or stable.…”

“…This data will be valuable for the development of new computational models of cell migration and adhesion. In fact, we have already used the data to develop a new model that has helped us to further understand the role of paxillin S273 phosphorylation and its effects on the small GTPases Rac1 and RhoA in regulating adhesion dynamics 48 . In this study, we identified two paxillin-WT adhesion subpopulations experimentally: one was small dynamic adhesions and one was larger stable adhesions 48 .…”

“…In fact, we have already used the data to develop a new model that has helped us to further understand the role of paxillin S273 phosphorylation and its effects on the small GTPases Rac1 and RhoA in regulating adhesion dynamics 48 . In this study, we identified two paxillin-WT adhesion subpopulations experimentally: one was small dynamic adhesions and one was larger stable adhesions 48 . This bimodality was abolished experimentally with expression of paxillin-S273D or S273A mutants, which had predominantly small, dynamic or large, stable adhesions, respectively 48 .…”

LncRNA CDKN2B-AS1/miR-141/cyclin D network regulates tumor progression and metastasis of renal cell carcinoma

“…Elevated paxillin phosphorylation is common in cancer tissues and is associated with tumorigenesis, EMT, and metastasis 15,16,50 . Paxillin interacts with numerous molecules, controlling the Rho family of GTPases, that are crucial regulators of adhesion dynamics 16,51 . Rac1 GTPase is required for cell migration and its hyperactivation results in cancer invasiveness and progression 52 .…”

Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration

“…As in the previous model, we used quantitative mass spectrometry data to determine the prevailing protein isoforms of the RhoA-Rac1 network in MDA-MB-231 cells, as well as the protein abundances. The parameters of activation and deactivation of RhoA, Rac1 and PAK were estimated based on the literature data ( Lyda et al, 2019 ; Tang et al, 2018 ). The parameters of activation and deactivation of DIA and ROCK were estimated based on typical association and dissociation constants of protein-protein interactions ( Kholodenko et al, 1999 ).…”