Protein phosphorylation, the process by which proteins are flipped from one activation state to another, is a crucial function for most living beings, since it controls nearly every cellular process, including metabolism and gene transcription.

Biologists have learned that interactions by kinases (enzymes that add a phosphate to a protein) are highly regulated. Each of the 428 human serine/threonine kinases interact only with certain substrate proteins, and they pick their "partners" unfailingly. But for the reverse reaction, called dephosphorylation (removing a phosphate from a protein), only about 40 phosphatases are available to interact with all substrate proteins. In fact, just one of them, protein phosphatase 1 (PP1), is believed to be responsible for up to 65 percent of dephosphorylation reactions.

New research published in Nature Structural and Molecular Biology reveals that PP1 "chooses" proteins in dephosphorylation reactions based on which of its binding sites is available for the interaction to occur. The finding is important, because erroneous PP1 regulation can cause numerous diseases, including cancer (chromatin remodeling), diabetes (glycogen) and Parkinson's (LTP).


the phosphate spinophilin binds to one of PP1's three available binding sites, reducing the number of available substrates for other proteins.

(Photo Credit: Wolfgang Peti, Brown University)

The team showed for the first time how PP1 is bound to a regulator protein, spinophilin. Using nuclear magnetic resonance spectroscopy and X-ray crystallography, the researchers examined the spinophilin-PP1 complex's structure and saw that spinophilin had attached itself to one of three substrate binding sites on PP1, called the C-terminal substrate binding groove.

Examined at the atomic scale, it appears as if spinophilin is a many-tentacled beast that has woven itself into the C-terminal substrate binding groove, effectively blocking any substrate requiring this groove from interacting with PP1.

That leaves only two other binding sites, the acidic and hydrophobic substrate binding grooves. "Any substrate that needs the C-terminal is out of the game," said Wolfgang Peti, Assistant Professor of Medical Science at Brown University.

By narrowing the binding sites from three to two, PP1 is in effect becoming more selective, Peti noted. "What that means is PP1 is equally tightly controlled as kinases are," he said.

"Now we know how PP1 is regulated," Peti added. "What simply happens is we don't create more enzymes. We create more protein complexes (holoenzymes) that increase the specificity of PP1."



Citation: Ragusa et al., 'Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites', Nature Structural&Molecular Biology, March 2010; doi:10.1038/nsmb.1786 Article