Abstract
Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.
Highlights
Protein phosphorylation is a ubiquitous mechanism of signal transduction that is regulated by the opposing actions of kinases and phosphatases [1,2]
Multisite z-Chain Phosphorylation z-chains with all combinations of immunoreceptor tyrosine-based activation motifs (ITAMs) mutants, we found no evidence for sequential phosphorylation, and surprisingly, we found that phosphorylation sites did not modulate the switchlike response but did modulate receptor potency and maximum phosphorylation
We examined the contribution of each ITAM to the ZAP-70-mediated increase in potency observed for the wild-type z-chain
Summary
Protein phosphorylation is a ubiquitous mechanism of signal transduction that is regulated by the opposing actions of kinases and phosphatases [1,2]. A well-known regulatory mechanism is a switchlike response, whereby the phosphorylation state of a protein can be highly sensitive to the concentrations of its modifying kinase and phosphatase. Mathematical models have shown that switchlike responses can be produced by a number of mechanisms that rely on multiple phosphorylation sites, such as substrate sequestration [8], nonessential sites [9], local concentrations [10], independent binding sites [11], membrane-anchoring (diffusion-limited reactions) [12], entropic mechanisms [13], binding of effector molecules [14], cooperativity between sites, or site-specific enzymatic rates [15], and other mechanisms reviewed elsewhere [2,6,7]. There are few experimental studies aimed at a systematic investigation of multisite phosphorylation
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