Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases.

The reversible phosphorylation of proteins is central to the regulation of most aspects of cell function but, even after the first protein kinase was identified, the general significance of this discovery was slow to be appreciated. Here I review the discovery of protein phosphorylation and give a personal view of the key findings that have helped to shape the field as we know it today.

The origins of protein phosphorylation

Understanding the regulation of hepatic glucose metabolism had its foundation in the elucidation of several pathways, but recent advances have come from the application of molecular genetics. Five years ago little was known about the primary structure of the key regulatory enzymes. Since then, the primary sequence of liver GK, 6-PF-1-K, Fru-1,6-P2ase, PK, PEPCK, and 6-PF-2-K/Fru-2,6-P2ase have been derived from cDNA sequences and/or determined by direct protein sequencing. This has provided new insights into the molecular mechanisms of catalysis and the regulation of these enzymes by covalent modification. Isolation of the cDNAs for these enzymes also has allowed for the quantitation of specific mRNAs and permitted analysis of hormonal control of specific gene expression. The genes for these enzymes have been isolated and sequenced, and their promoter regions are being identified and characterized. Hormone response elements have been delineated in several of the promoters. The promoter regions for 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase have also been identified, and future research will focus on the elucidation of the mechanisms whereby hormones regulate the expression of these genes. A number of generalizations can be made about the regulation of gene expression of glycolytic/gluconeogenic enzymes. First, there is coordinate hormonal regulation of gene expression and these effects are consonant with their physiologic actions. Insulin induces the mRNAs that encode glycolytic enzymes and represses the mRNAs that encode gluconeogenic enzymes; cAMP has opposite effects. Both can increase or decrease transcription. Whereas insulin and cAMP affect all of these mRNAs, glucocorticoids appear to have a more restricted action. Second, transcriptional and posttranscriptional regulatory mechanisms are involved. The synthesis of all of the mRNAs discussed is regulated by hormones. Relatively little is known about how mRNA stability is regulated in general, but it is clear that PEPCK mRNA is stabilized by agents that increase the rate of transcription of the gene. Under appropriate metabolic signals this dual control of mRNA synthesis and stability provides for a long-term increase in PEPCK mRNA and protein. Studies with PK mRNA are less direct, but suggest a similar dual mechanism. It will be interesting to see whether multilevel regulation is restricted to these two mRNAs, both of which are involved in the same substrate cycle, or whether the stability of other mRNAs involved in hepatic glucose metabolism is also affected. Third, glucose appears to be important in the regulation of these hepatic genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.

Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase.

https://faculty.washington.edu/scottjdw/pdfs/scott_pharmathera_1991_p123.pdf

Cyclic nucleotide-dependent protein kinases

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver.

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase

Proteolytic modification of pig and rat liver pyruvate kinase type L including phosphorylatable site.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2875%2981111-2

Subtilisin-catalyzed removal of phosphorylated site of pig liver pyruvate kinase without inactivation of the enzyme

Rat-liver cell sap protein was separated into four fractions by pH 5.5 precipitation and ammonium sulfate fractionation. On incubation with (32P)ATP, protein of two of the fractions was phosphorylated, showing the presence of protein kinase activity and endogenous protein substrates. The phosphorylation of one of the fractions was markedly stimulated by cyclic 3′,5′-AMP. Strong evidence was obtained that this phosphate-incorporating material was neither active phosphorylase, phosphorylase kinase nor glycogen synthetase. The two phosphate-incorporating fractions were chromatographed on DEAE-cellulose with stepwise elution. Several subtractions contained material which was phosphorylated on incubation with (32P)ATP. The phosphorylation of two subfractions was greatly stimulated by cyclic 3′,5′-AMP. By polyacrylamide gel electrophoresis in detergent of (32P)ATP-incubated material from these fractions, three major 32P-labelled components with estimated molecular weights of 62000, 55000 and 47 000 were demonst...

Elisabeth Humble

Papers

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver.

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase

Proteolytic modification of pig and rat liver pyruvate kinase type L including phosphorylatable site.

Subtilisin-catalyzed removal of phosphorylated site of pig liver pyruvate kinase without inactivation of the enzyme

Cyclic 3',5'-AMP-stimulated and non-stimulated phosphorylation of protein fractions from rat-liver cell sap on incubation with (gamma-32P)ATP.