Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Abstract

Caged phosphopeptides and phosphoproteins are valuable tools for dissecting the dynamic role of phosphorylation in complex signaling networks with temporal and spatial control. Demonstrating the broad scope of phosphoamino acid caging for studying signaling events, we report here the semisynthesis of a photolabile precursor to the cellular migration protein paxillin, which is a complex, multidomain phosphoprotein. This semisynthetic construct provides a powerful probe for investigating the influence that phosphorylation of paxillin at a single site has on cellular migration. The 61-kDa paxillin construct was assembled using native chemical ligation to install a caged phosphotyrosine residue at position 31 of the 557-residue protein, and the probe includes all other binding and localization determinants in the paxillin macromolecule, which are essential for creating a native environment to investigate phosphorylation. Following semisynthesis, paxillin variants were characterized through detailed biochemical analyses and by quantitative uncaging studies.

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1

“…In contrast, tyrosine 181 seemed irrelevant. These results are consistent with requirements for paxillin tyrosine 31 and 118 phosphorylation for adhesion, spreading, and migration in other systems [14,45,46]. Serine phosphorylation of paxillin may also influence epithelial adhesion [47,48], and our studies of paxillin tyrosine phosphorylation do not preclude the possibility that pressure may also stimulate serine phosphorylation.…”

Chemoselektive Ligations‐ und Modifikationsstrategien für Peptide und Proteine

“…[3,9,[279][280][281] [74,282,283] Acetylierungen oder Methylierungen, [105] Glycosylierungen [284,285] und Lipidierungen [286] sowie auch nichtnatürliche Modifikationen oder Aminosäuren wie Aminophenylalanin, [287] Fluortyrosin, [288] ATP-verknüpftes Phenylalanin, [289] d-Aminosäuren [290] oder Phosphoaminosäuremimetika [291] in Proteine integriert werden. Weiterhin ist die Installation von Fluorophoren und Affinitätsmarkern in definierter und homogener Weise möglich, was ein entscheidender Vorteil gegenüber konventionellen Proteinmarkierungsmethoden ist, die häufig eine geringere Spezifität aufweisen.…”

“…[298] Die Proteinkinasen, die diese Phosphorylierungen vermitteln, sind häufig unbekannt oder biochemisch nicht zugänglich sind, weshalb man die Proteinsemisynthese als eine wichtige Methode etabliert hat, um selektive Phosphorylierungen vorzunehmen. [283,299] Die Proteinsemisynthese kann auch genutzt werden, um chemische Gruppierungen einzufügen, die eine selektive Maskierung und Demaskierung der Proteinphosphorylierung ermögli-chen.…”

Semisynthetic Enzymes

“…Kent et al introduced the first method for native chemical ligation (NCL) to couple unprotected peptide fragments in solution through native peptide bond formation at a ligation site [126]. NCL is well suited for production of semisynthetic proteins [127]. The trans-thioesterification reaction takes place between the thiol group of N-terminal Cys of one peptide and a C-terminal thioester moiety of a second peptide.…”