Phosphorylase and the Origin of Reversible Protein Phosphorylation Prof. Edmond Fischer

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1 hosphorylase and the Origin University of Washington, Seattle, USA 1 55 years ago Endocrinology was well-established, but remained in the phenomenological level Insulin was known as the message sent by the pancreas, epinephrine by the adrenal Nobody knew how these hormones acted Discovery of cam secondary messenger One observes a physiological phenomena, and then tries to find the factors responsible for it (Claude Bernard) Today it is the other way around We went from 6 functions in search of an enzyme, to thousands of proteins and enzymes, in search of a function 2 Edwin G. Krebs passed away on December , two months after this lecture was recorded 3 1

2 Degradation of glycogen Glycogen hosphorylase Glucose-1- + Adrenaline Blood Glucose (liver) Energy: AT (in all tissues) 4 5 Degradation of glycogen Glycogen synthase Glycogen + Adrenaline Insulin hosphorylase Glucose-1- + Glucagon Blood Glucose (liver) Energy: AT (in all tissues) 6 2

3 7 Degradation of glycogen Blood Glucose (liver) Glycogen +i Glucose-1- hosphorylase Energy: AT (in all tissues) The action of phosphorylase The deposition of glucose in glycogen is thermodynamically preferred However, the ratio of inorganic phosphate to G-1- was too lopsided The role of AM in the activity of phosphorylase 8 Regulation of phosphorylase (Cori & Cori ) AM Inactive Active AM AM + b AM a 9 3

4 10 The assumption was that AM served as a co-enzyme We had isolated phosphorylase from potatoes and showed that there was no requirement for AM We knew that co-enzymes were conserved among species

5 Cori s preparation of muscle phosphorylase ca Replacing the filter paper with centrifugation We could never obtain active phosphorylase

6 Cori s preparation of muscle phosphorylase Muscle hosphorylase b We could not believe that we were dealing with real phosphorylase h b hosphorylase a Cori s preparation of muscle phosphorylase Muscle hosphorylase b p At first, we did not know what was phosphorylated A protein that could be precipitated by TCA hosphorylase AT AT hosphorylase a 18 6

7 Activation-inactivation cycle of phosphorylase hosphorylase Kinase Mg 2+ AT i hosphorylase 'ase hosphorylase b (inactive) hosphorylase a (active) Activation-inactivation cycle of phosphorylase hosphorylase Kinase Mg 2+ AT hosphorylase b (inactive) i hosphorylase 'ase hosphorylase a (active) 21 7

8 Activation-inactivation cycle of phosphorylase hosphorylase Kinase Mg 2+ AT i hosphorylase b (inactive) hosphorylase 'ase hosphorylase a (active) 22 Hormonal control of glycogenolysis A cascade of two enzymes working on one another, both activated by a phosphorylation reaction Inorganic phosphate was released when active phosphorylase a was converted to the b form Discovery of cam phosph. kinase phosph. kinase Mg AT phosph. b phosph. a glycogen + i glucose

9 Hormonal control of glycogenolysis Hormone Adenylate Cyclase AT cam cam-dep rot. kinase cam-dep rot. kinase Kinase kinase When will it stop? phosph. kinase phosph. kinase Mg AT phosph. b phosph. a glycogen + i glucose-1 25 Nerve Impulse Muscle thin filament Actin-Myosin interaction CONTRACTION

10 28 29 We did not know if the phosphorylation reaction was restricted to the activation of phosphorylase erhaps regulation by yphosphorylation p is restricted to the enzymes of glycolysis? Nerve Impulse hosphorylase kinase Muscle thin filament hosphorylase Actin-Myosin interaction GLYCOLYSIS CONTRACTION AT 30 10

11 Ubiquitous protein phosphorylation Cell death Signal Transduction Metabolism Cell Cycle Transcription Transport rotein hosphorylation-h hosphorylation Dephosphorylation a Translation Cytoskeletal rearrangement Immune Response Secretion Transformation Differentiation 31 Regulation of phosphorylase Kinase hosphatase Inactive Active Occurs in a loose N-terminal arm, easily cleaved Simple to isolate the phospho-peptide A single serine residue had been phosphorylated 32 Reciprocal control of phosphorylase and glycogen synthase Kinase + hosphorylase Far more than one phosphate was introduced Glycogen (n) Glycogen (n-1) Glycogen Synthase Kinase 33 11

12 Control of glycogen synthesis and breakdown Glycogen cam-dep K hos Kinase /CaM dep K KC hos hos Glycogen Kinase + phorylase Synthase GSK-3 Casein Kinase 1 Casein Kinase 2 GSK-4 Glucose-1 34 Mechanism of action of GSK-3 We have been often asked if we realized at the beginning that we were working on a very ubiquitous reaction Absolutely not! Major phosphorylation sites CK-2 CK-1 N ~ 500 residues a b c d a b C hos K CK-1 Cam-K GSK-4 Ka GSK-3 CK-2 Adapted from eter Roach RASVSSLSRHSSHQSEDEEE

13 37 Before After 38 Thank you for listening 39 13

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