Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and phar-macological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenome-nological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 trans -membrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

Role of the membrane anchor in the regulation of Lck activity / Porciello, Nicla; Cipria, Deborah; Masi, Giulia; Lanz, Anna-Lisa; Milanetti, Edoardo; Grottesi, Alessandro; Howie, Duncan; Cobbold, Steve P; Schermelleh, Lothar; He, Hai-Tao; D'Abramo, Marco; Destainville, Nicolas; Acuto, Oreste; Nika, Konstantina. - In: JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 1083-351X. - 298:12(2022). [10.1016/j.jbc.2022.102663]

Role of the membrane anchor in the regulation of Lck activity

Porciello, Nicla;Masi, Giulia;Milanetti, Edoardo;Grottesi, Alessandro;D'Abramo, Marco;Acuto, Oreste
;
2022

Abstract

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and phar-macological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenome-nological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 trans -membrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.
2022
CD45; Lck; boundary lipids; membrane anchor; membrane lateral organization
01 Pubblicazione su rivista::01a Articolo in rivista
Role of the membrane anchor in the regulation of Lck activity / Porciello, Nicla; Cipria, Deborah; Masi, Giulia; Lanz, Anna-Lisa; Milanetti, Edoardo; Grottesi, Alessandro; Howie, Duncan; Cobbold, Steve P; Schermelleh, Lothar; He, Hai-Tao; D'Abramo, Marco; Destainville, Nicolas; Acuto, Oreste; Nika, Konstantina. - In: JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 1083-351X. - 298:12(2022). [10.1016/j.jbc.2022.102663]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1667891
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