Press Archive

A New Kind of Deregulation: UCSD/SDSC Researchers Find Clues to Cellular Signal System

Published 08/10/1995

In living organisms, processes including cell growth and division, tissue differentiation in embryos, memory and neurotransmission, and most of the complex instructions for cellular changes from birth to death are regulated by a family of enzymes called the protein kinases. Faulty regulation of such kinases is involved in or can cause many diseases, such as diabetes and cancer. Now, scientists from UCSD and the San Diego Supercomputer Center (SDSC) have taken major steps in understanding these enzymes and their activities.

In articles appearing today in the scientific journals Cell and Science, the researchers give a three-dimensional description of the processes, which are templates for the rational design of drugs or pharmaceuticals capable of regulating the intricate intracellular signaling system for clinical applications. They also identified a novel targeting signal. The work points to ways to attack a variety of human ailments.

"All these protein kinases have to be kept off when you don't need them," said Susan Taylor, UCSD chemist and Senior Fellow at SDSC, who is the principal investigator for the two studies. "So a major way to regulate them is to inhibit their activity--turn them off--until needed, or to move them to a different part of the cell."

"Now that the basic structures and the function of one of these kinases and its inhibitor are known in detail, we can seriously begin to design some better inhibitors," she said.

Any highly evolved cell--plant, animal, or human--can be thought of as a massive dispatch station or telephone switchboard. Instead of voice, paper, or electronic signals, however, this system passes molecular messages back and forth in relays that circulate the signals from one cellular structure to another, from the nucleus to various places in the cytoplasm to (or through) the outer cell membrane and back.

One of the principal signaling systems is a constant dance of phosphorylation and dephosphorylation, adding highly charged phosphate groups to (or deleting them from) signaling molecules. The presence or absence of the phosphate groups changes the structure and function of the modified molecules--often other kinase molecules. Details of such a system are explained for the first time in the two articles appearing today.

"What we have found are fundamental steps in the path along which kinases are activated, targeted to some part of the cell, and deactivated," said Lynn Ten Eyck of SDSC, coauthor of the Science paper, who codirects the Computational Center for Macromolecular Structure (CCMS) with Taylor. CCMS is an NSF-funded project at SDSC, under which the computation for the work reported was carried out.

When a signal, such as a hormone, binds to the outside of the cell, the cell must translate that signal into a biological response that causes the protein kinases to get busy inside. The "first messenger" to the cell is the hormone, and its binding signals the synthesis of a second messenger, a small molecule inside the cell.

The kinase family contains several hundred distinct members, ranging from the simple protein kinase A (PKA), studied by the researchers, to more and more complex or specific kinases. For PKA, the first messenger is frequently the hormone adrenaline. The second messenger, inside the cell, is cyclic adenosine monophosphate (cyclic AMP or cAMP), which is a ubiquitous second messenger in all cells.

In the inactive state, PKA consists of two catalytic subunits (C) and two regulatory subunits (R). The separation of R from C, which is the key activation step, is triggered by the binding of cAMP to the R subunits. When cAMP molecules bind to the R subunits, the PKA splits into three pieces--the two R subunits and cAMP molecules stay together, and the two C subunits are each freed to interact with other proteins.

In the Science study, Taylor's group examined a mutant but functional form of a single R subunit of PKA. Their studies revealed the three-dimensional structure of the R subunit and the two sites (A and B) where cAMP binds to R--the "cAMP binding domains."

The single R units were crystallized and their structure studied by X-ray crystallography (both a multiwire area detector at UCSD and a synchrotron at Stanford were used) in combination with computational refinement and modeling, some of which required the aid of a large supercomputer (Cray C90) at SDSC. Subsequent scientific visualization of the structure showed the specific topography of each cAMP binding domain as an arrangement of amino acids resembling a "jelly roll," according to Ying Su, a graduate student of Taylor's and the paper's first author. The work constitutes her Ph.D. dissertation.

The subtlety here is in the order of binding. The first cAMP molecule binds to the B site, and this changes the shape or conformation of the R subunit. The structural change sets the stage for easier binding by a second cAMP molecule, this time to the A site. Although this "cooperative binding" was known to occur, the reasons for it, which have to do with the conformational change in the structure of the R unit, were only revealed by this research.

The other authors of the Science article include Taylor, Ten Eyck, Wolfgang Dostmann (Institute for Pharmacology, University of Munich), Friedrich Herberg (University of the Ruhr at Bochum), Kyle Durick (postdoctoral researcher in Taylor's group), Nguyen-hu Xuong (UCSD Departments of Biology, Chemistry, and Physics; inventor of the multiwire device), and K.I. Varughese (a Senior Researcher in Xuong's lab at UCSD).

Turning back to an examination of the catalytic (C) subunit of PKA, Taylor and colleagues have also published a major paper in this week's Cell, a prestigious biological journal. The original solution of the C subunit by the Taylor group in 1991 (Science v.263, p.407) was of a complex consisting of the C subunit and part of a well-known inhibitor of C's activity, called protein kinase inhibitor (PKI). By using fluorescent tags for parts of this inhibitor, the group demonstrated in laboratory experiments that a very small sequence of amino acids embedded in PKI constitutes a signal that instructs the C subunit to leave the cell nucleus and move out into the cytoplasm, where it becomes available for binding to an R subunit.

This nuclear export signal (NES) is the first such signal identified. Scientists have discovered a number of nuclear localization signals (NLS) which translocate molecules containing an NLS into the cell nucleus, but there has been debate about whether a clear signal exists to tell compounds to leave the nucleus. The Cell paper's first author is Wei Wen (graduate student in the Taylor group), who also showed that a similar signal is found in other proteins, such as HIV-Rev, a protein essential for biosynthesis of the active HIV virus.

Other Cell authors include Taylor, Judy Meinkoth (Department of Pharmacology, University of Pennsylvania), and Roger Y. Tsien (Department of Chemistry and Biochemistry, UCSD, and Department of Pharmacology and Howard Hughes Medical Institute, UCSD School of Medicine).

The work in the Science paper was supported by the National Institutes of Health (NIH), the Lucille P. Markey Charitable Trust, and the National Science Foundation. The work in the Cell paper was funded by NIH and its National Institute of Diabetes and Digestive and Kidney Diseases, and by the California Tobacco-Related Disease Research Program.

The San Diego Supercomputer Center, a national laboratory for computational science and engineering, is sponsored by the National Science Foundation, administered by General Atomics, and affiliated with the University of California at San Diego. For additional information, see http://www.sdsc.edu or contact Ann Redelfs, SDSC, redelfs@sdsc.edu, 619-534-5032.

References:

Su, Y., W.R.G. Dostmann, F.W. Herberg, K. Durick, N-h. Xuong, L. Ten Eyck, S.S. Taylor, K.I. Varughese: Regulatory subunit of protein kinase A: Structure of deletion mutant with cAMP binding domains, Science 269, 807-819 (August 11, 1995).

Wen, Wei, Judy L. Meinkoth, Roger Y. Tsien, and Susan S. Taylor: Identification of a signal for rapid export of proteins from the nucleus, Cell 82, 1-20 (August 11, 1995).


For more information, contact:

Ann Redelfs, SDSC
(619) 534-5032
redelfs@sdsc.edu

Warren Froelich, UCSD
(619) 534-8564
wfroelich@ucsd.edu

Merry Maisel, SDSC
(619) 534-5127
maisel@sdsc.edu