Genetic Encoding of Phosphorylated Amino Acids into Proteins.

Reversible phosphorylation is a fundamental mechanism for controlling protein function. Despite the critical roles phosphorylated proteins play in physiology and disease, our ability to study individual phospho-proteoforms has been hindered by a lack of versatile methods to efficiently generate homogeneous proteins with site-specific phosphoamino acids or with functional mimics that are resistant to phosphatases. Genetic code expansion (GCE) is emerging as a transformative approach to tackle this challenge, allowing direct incorporation of phosphoamino acids into proteins during translation in response to amber stop codons. This genetic programming of phospho-protein synthesis eliminates the reliance on kinase-based or chemical semisynthesis approaches, making it broadly applicable to diverse phospho-proteoforms. In this comprehensive review, we provide a brief introduction to GCE and trace the development of existing GCE technologies for installing phosphoserine, phosphothreonine, phosphotyrosine, and their mimics, discussing both their advantages as well as their limitations. While some of the technologies are still early in their development, others are already robust enough to greatly expand the range of biologically relevant questions that can be addressed. We highlight new discoveries enabled by these GCE approaches, provide practical considerations for the application of technologies by non-GCE experts, and also identify avenues ripe for further development.

Preparation and application of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies.

This unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non-purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence-specific anti-phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross-reactivity of such antibodies, which is a common problem with anti-peptide antibodies, is also discussed.

Adhesion-associated and PKC-modulated changes in serine/threonine phosphorylation of p120-catenin.

p120-catenin (p120) was originally identified as a tyrosine kinase substrate, and subsequently shown to regulate cadherin-mediated cell-cell adhesion. Binding of the p120 Arm domain to E-cadherin appears to be necessary to maintain adequate cadherin levels for strong adhesion. In contrast, the sequence amino-terminal to the Arm domain confers a negative regulatory function that is likely to be modulated by phosphorylation. Several agents that induce rapid changes in cell-cell adhesion, including PDBu, histamine, thrombin, and LPA, result in significant changes in p120 S/T phosphorylation. In some cases, these changes are PKC-dependent, but the relationship among adhesion, PKC activation, and p120 phosphorylation is unclear, in part because the relevant p120 phosphorylation sites are unknown. As a crucial step toward directly identifying the function of these modifications in adhesion, we have used two-dimensional tryptic mapping and site-directed mutagenesis to pinpoint the constitutive and PKC-modulated sites of p120 S/T phosphorylation. Of eight sites that have been identified, two were selectively phosphorylated in vitro by GSK3 beta, but in vivo treatment of cells with GSK3 beta inhibitors did not eliminate these sites. PKC stimulation in vivo induced potent dephosphorylation at S268, and partial dephosphorylation of several additional sites. Surprisingly, PKC also strongly induced phosphorylation at S873. These data directly link PKC activation to specific changes in p120 phosphorylation, and identify the target sites associated with the mechanism of PKC-dependent adhesive changes induced by agents such as histamine and PDBu.