Structural basis for control by phosphorylation.

Posttranslational modification by phosphorylation is a ubiquitous regulatory mechanism in both eukaryotes and prokaryotes. Intracellular phosphorylation by protein kinases, triggered in response to extracellular signals, provides a mechanism for the cell to switch on or switch off many diverse processes. These processes include metabolic pathways, kinase cascade activation, membrane transport, gene transcription, and motor mechanisms. The reverse reaction of dephosphorylation is catalyzed by protein phosphatases that are controlled by response to different stimuli so that phosphorylation and dephosphorylation are separately controlled events. In eukaryotes, the protein kinase domain responsible for phosphorylation on serine, threonine, or tyrosine residues is the first, second, and third most common domain in the genome sequences of yeast (S. cerevisiae), the worm (C. elegans), and the fly (D. melanogaster), respectively, indicating the importance of phospho-signaling in higher organisms. The human genome contains 575 eukaryotic protein kinase domains, representing 2% of the total genome and the third most populous domain.1 In prokaryotes, signaling by phosphorylation is equally important. In E. coli there are 62 genes that encode proteins involved in dual histidine kinase/response regulator systems, representing approximately 1.5% of the entire genome. In this review we ask what are the structural consequences of phosphorylation and how is this structural response correlated with signal? We limit the examples for the most part to those where the structures of both the phosphoand non-phospho protein are known and coordinates available in the Protein Data Bank. We also limit our examples to phosphorylation of intact proteins and exclude the numerous structural examples of phospho-peptide binding to cognate proteins such as phospho-tyrosine peptide binding to SH2 (e.g., ref 2) and PTB domains (e.g., ref 3) (reviewed in ref 4) and phospho-serine peptide binding to 14-3-3 domains.5 The phosphopeptide interactions give insights into the recognition by other proteins of the phospho-amino acid, an important result especially for the design of ligands that might interfere with this association, but these structures do not illuminate how the recognition site is engineered in the target protein as a consequence of phosphorylation nor the response of the phosphoprotein on recognition. We also exclude results where a phospho-protein is an intermediate in a reaction pathway such as in the phosphatases,6,7 topoisomerases,8 and Cre recombinase.9 In these examples, phosphorylation is part of the catalytic pathway and does not result in a response in the target protein that signals to other proteins. Finally, phenylalanine hydroxylase is an allosteric protein regulated by phenylalanine, tetrahydrobiopterin, and phosphorylation on serine 16. The non-phospho and phospho * To whom correspondence should be addressed. Phone: 01865 275365. Fax: 01865 510454. E-mail: [email protected]. 2209 Chem. Rev. 2001, 101, 2209−2242