Reversible Phosphorylation and Kinase Cascades: the Work of Edwin G. Krebs

Abstract

An Adenosine 3′,5′-Monophosphate-dependant Protein Kinase from Rabbit Skeletal Muscle (Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968) J. Biol. Chem. 243, 3763–3765) Multiple Components in an Epidermal Growth Factor-stimulated Protein Kinase Cascade. In Vitro Activation of a Myelin Basic Protein/Microtubule-associated Protein 2 Kinase (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220–4227) Edwin Gerhard Krebs was born in Lansing, IA in 1918. In 1936, he entered the University of Illinois planning to major in a branch of science related to chemistry. By the beginning of his 4th year in college, he had narrowed his choices down to either getting an advanced degree in organic chemistry or going to medical school. After receiving a scholarship to attend the Washington University School of Medicine in St. Louis, he decided to become a physician. However, during his 4th year at the University of Illinois he carried out undergraduate research in organic chemistry and found it to be a fascinating experience. Although he continued on to medical school, this introduction to research influenced his medical training and was a strong factor in his eventual decision to become a research biochemist rather than a clinician. The Washington University School of Medicine proved to be a place where Krebs could receive classical medical training and also participate in medical research. He undertook several research projects, first with Philip A. Schafer and later with Arda A. Green. It was during this period that Krebs first heard about phosphorylase, which was crystallized by Green and Carl and Gerty Cori, as reported in a previous Journal of Biological Chemistry (JBC) Classic (1Cori C.F. Cori G.T. JBC ClassicsJ. Biol. Chem. 1928; 79: 321-341Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1938; 124: 543-555Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1939; 127: 771-782Abstract Full Text PDF Google ScholarGreen A.A. Cori G.T. J. Biol. Chem. 1943; 151: 21-29Abstract Full Text PDF Google ScholarCori G.T. Green A.A. J. Biol. Chem. 1943; 151: 31-38http://www.jbc.org/cgi/content/full/277/29/e18Abstract Full Text PDF Google Scholar). Green and the Coris found that phosphorylase exists in two forms, phosphorylase a, which was active without the addition of AMP, and phosphorylase b, which was inactive without AMP. After graduating from medical school in 1943, Krebs completed an 18-month residency in internal medicine at Barnes Hospital in St. Louis and then became a medical officer in the navy. He was discharged in 1946 and returned to St. Louis with the idea of continuing his training in internal medicine. However, a 2-year-long waiting list for the residency program caused him instead to begin studying science. Because of his background in chemistry he chose to study biochemistry and was accepted by the Coris as a postdoctoral fellow. Krebs worked with the Coris for 2 years, investigating the interaction of protamine with rabbit muscle phosphorylase. He found the work so rewarding that he decided to continue doing research rather than returning to internal medicine. In 1948, Krebs was invited to become an assistant professor of biochemistry at the University of Washington School of Medicine. Krebs had been in Seattle for 5 years when Edmond H. Fischer joined the department. Fischer had received his doctorate from the University of Geneva in 1947 studying potato phosphorylase. Because American universities offered more opportunities in the new field of biochemistry, Fischer moved to the United States to do a postdoctoral fellowship at the California Institute of Technology. He was then offered an assistant professorship at the University of Washington where he began a long association with Krebs. Together Krebs and Fischer decided to see if they could determine the mechanism by which the inactive phosphorylase b was converted to phosphorylase a. Based on the work of the Coris, Krebs and Fischer believed that a prosthetic group, which was some form of AMP, was involved in the activation of phosphorylase b. However, they soon discovered that the interconversion of phosphorylase was the result of an enzyme-catalyzed phosphorylation-dephosphorylation reaction. Similar work was being carried out on liver phosphorylase at approximately the same by Earl Sutherland. As discussed in a previous JBC Classic (2Rall T.W. Sutherland E.W. JBC ClassicsJ. Biol. Chem. 1958; 232: 1065-1076Abstract Full Text PDF PubMed Google ScholarSutherland E.W. Rall T.W. J. Biol. Chem. 1958; 232: 1077-1092http://www.jbc.org/cgi/content/full/280/42/e39Abstract Full Text PDF PubMed Google Scholar), Sutherland discovered the second messenger cyclic AMP (cAMP), which he showed promoted the phosphorylation and activation of phosphorylase. The way in which cAMP promoted phosphorylase activation was eventually elucidated when Krebs and Fischer discovered phosphorylase kinase, which was responsible for phosphorylating phosphorylase. Phosphorylase kinase itself existed in a highly activated phosphorylated form and a less active nonphosphorylated form. Fischer and Krebs continued to work on their own specific areas related to phosphorylase. One of Krebs' projects was concerned with the molecular mechanism of action of cAMP in promoting phosphorylase activation via phosphorylase kinase. This was eventually determined when Krebs' postdoctoral fellow Donal A. Walsh discovered the cAMP-dependent protein kinase (PKA), which is the subject of the first JBC Classic reprinted here. Using rabbit skeletal muscle, Krebs, Walsh, and John P. Perkins isolated a protein kinase that activated phosphorylase kinase and was completely dependent on cAMP for its activity. Because PKA catalyzed the phosphorylation of proteins other than phosphorylase kinase, they proposed a general name rather than calling the new enzyme phosphorylase kinase kinase. The discovery of PKA established the existence of the first protein kinase cascade in which one kinase activates another kinase. It also stimulated work on the protein phosphorylation process in general. Soon it became evident that reversible protein phosphorylation was a fundamental biological mechanism. By the 1970s biochemical research on protein phosphorylation was so extensive that 5% of papers in biology journals dealt with the subject. As a result of the significance of their discovery, Krebs and Fischer were awarded the 1992 Nobel Prize in Physiology or Medicine “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism.” In 1968 Krebs left the University of Washington to assume the position of founding chairman of the Department of Biological Chemistry at the University of California, Davis. He stayed in California for 8 years and continued to work on PKA. Krebs then returned to the University of Washington as chairman of the Department of Pharmacology. He was also a Howard Hughes Medical Institute Investigator from 1977 to 1990. Currently, he is a Professor Emeritus in the Department of Biochemistry at the University of Washington. During this second period at the University of Washington, Krebs became interested in protein tyrosine phosphorylation and the mechanism of action of growth factors. In the 1970s it was established that proteins can be phosphorylated on tyrosine as well as on serine and threonine and that protein-tyrosine kinases (PTKs) often acted as receptors for growth factors. Because many cellular responses to growth factors whose receptors were PTKs were mediated by changes in serine/threonine phosphorylation, Krebs wanted to know how protein tyrosine phosphorylation was coupled to protein serine/threonine phosphorylation. He approached the problem by working upstream from the effect of growth factors on the phosphorylation of the ribosomal protein S6, which is phosphorylated on serine residues in response to PTK receptors. Krebs showed that a growth factor-stimulated mitogen-activated protein (MAP) kinase could phosphorylate and activate an S6 kinase. MAP kinase itself appeared to be activated by serine/threonine phosphorylation, suggesting the existence of a third protein-serine/threonine kinase in this growth factor-stimulated process. In the second JBC Classic reprinted here, Krebs, along with Natalie Ahn, demonstrates the existence of two separable activating factors that catalyze the activation of an inactive form of MAP kinase. Eventually these activating factors were shown to be MAP kinase kinases. Based on this research, Krebs established what is now known as the MAP kinase cascade. Recognition for Krebs' work on phosphorylation came from various awards and honors in addition to the Nobel Prize. He received the Whitaker Foundation's George W. Thorn Award (1983), the American Heart Association's Research Achievement Award (1987), the FASEB 3M Award (1989), the Albert Lasker Award for Basic Medical Research (1989), and the Robert A. Welch Award in Chemistry (1991). Krebs was also president of the American Society for Biological Chemists in 1985 and was an Associate Editor for the JBC from 1972 to 1993 and a member of the JBC editorial board from 1965 to 1970. He was also elected to the National Academy of Sciences and the American Academy of Arts and Sciences. 1All biographical information on Edwin G. Krebs was taken from Refs. 3Krebs E.G. Edwin G. Krebs — autobiography. Les Prix Nobel.in: Frängsmyr T. The Nobel Prizes 1992. Stockholm, 1993Google Scholar and 4Krebs E.G. An accidental biochemist.Annu. Rev. Biochem. 1998; 67: xiii-xxxiiCrossref Scopus (4) Google Scholar. 1All biographical information on Edwin G. Krebs was taken from Refs. 3Krebs E.G. Edwin G. Krebs — autobiography. Les Prix Nobel.in: Frängsmyr T. The Nobel Prizes 1992. Stockholm, 1993Google Scholar and 4Krebs E.G. An accidental biochemist.Annu. Rev. Biochem. 1998; 67: xiii-xxxiiCrossref Scopus (4) Google Scholar. Fischer also continues to do research at the University of Washington as an Emeritus Professor. He has received many honors for his scientific research over the course of his long career including election to the American Academy of Arts and Sciences and the National Academy of Sciences. Fischer was awarded the Werner Medal from the Swiss Chemical Society (1952), the Lederle Medical Faculty Award, the Prix Jaubert from the University of Geneva, and jointly with Krebs, the Senior Passano Award and the Steven C. Beering Award from Indiana University. 2All biographical information on Edmond H. Fischer was taken from Ref. 5Fischer E.H. Edmond H. Fischer — autobiography. Les Prix Nobel..in: Frängsmyr T. The Nobel Prizes 1992. Stockholm, 1993Google Scholar. 2All biographical information on Edmond H. Fischer was taken from Ref. 5Fischer E.H. Edmond H. Fischer — autobiography. Les Prix Nobel..in: Frängsmyr T. The Nobel Prizes 1992. Stockholm, 1993Google Scholar.