01 Jul 2005

Light switches come in two types, the on/off and the variable, or dimmer switch. Might phosphorylation switches on proteins work in two ways, also? While addition of a single phosphate can often turn a highly active protein into a dull, quiescent one, or vice versa, a report in today’s Science suggests that adding phosphates to different sites on the same protein might be the equivalent of adjusting the dimmer switch.

Barbara Graves and colleagues at the University of Utah, Salt Lake City, the University of British Columbia, Vancouver, and the University of Toronto write that variable phosphorylation “serves as a ‘rheostat’ for cell signaling to fine-tune transcription at the level of DNA binding.” The authors came to this conclusion after studying the transcription activator Ets-1. They also found that it was the flexible, relatively unstructured region of the protein that acts as the dimmer.

The findings suggest that protein phosphorylation be looked at in a new light, and hints of a new appreciation for multi-phosphorylated, random coil proteins, such as the microtubule binding protein tau, which forms the neurofibrillary tangles found in some Alzheimer disease brain tissue.

“We definitely think that the Ets-1 model could apply to other proteins,” said Graves, adding, “I think the potential of unstructured regions to do work is underappreciated. These territories tend to be less conserved, but have the benefit of being very accessible to enzymes that add post-translational modifications. Thus, these regions seem to be used to evolve new regulatory pathways, adding properties to proteins. Our findings on Ets-1 predict new types of regulation may be discovered in the analysis of other multiply modified proteins.”

Ets-1 contains an autoinhibitory module that flanks the DNA binding domain. In response to increases in intracellular calcium, variable phosphorylation of this module by CaM kinase II can reduce DNA binding affinity by up to 1,000-fold. To study this inhibition, first author Miles Pufall and colleagues mutated five kinase-sensitive serines in the serine-rich region (SRR) that plays a key role in the regulation.

Pufall and colleagues found that mutation of three of the five serines to alanine (S251A, S282A and S285A) significantly reduced inhibition of DNA binding. But they also discovered that multiple mutations produced a graded response. Conversion of one of these residues to alanine reduced the inhibitory effect of phosphorylation by about 20 percent, two mutations by about 12 percent, and if all three were mutated, phosphorylation of Ets-1 almost completely failed to prevent the factor from binding DNA. Pufall and colleagues also found that not all serines were created equal in this serine rich region. Phosphorylation of S285 dims DNA binding most, followed by that of S282 and S251.

Turning to the secondary structure, Pufall and colleagues used NMR analysis to show that the SRR has very little secondary structure and has amide hydrogen exchange rates similar to random coil protein. These rates of exchange are commonly used to identify how much of the protein is exposed to solvent, and thus predict how compact and convoluted the protein is.

All told, the data suggest that the consequences of multiple phosphorylation and flexible structure may be more profound than previously appreciated. In contrast to other proteins “…for which a threshold level of phosphorylation serves as a binary switch, multiple sites within Ets-1 regulate DNA binding in a graded manner across a side range of affinities, which is consistent with observed variable regulation in vivo,” write the authors. The also suggest that “the surprising use of a highly flexible segment in the graded regulation of Ets-1 DNA binding adds to the growing recognition of the role of unstructured protein regions in biology."—Tom Fagan

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