Chemical modification is a powerful tool for investigating the accessibility and function of specific amino acids within folded proteins. It has provided significant information regarding the role of different amino acids at the binding sites of numerous enzymes and DNA-binding proteins. The identification of such residues by chemical modification has then often be used to plan subsequent site-directed mutagenesis experiments. These data complement those from crystallographic and nuclear magnetic resonance (NMR) studies in determining the residues located at the active site; thus, one needs to consider all these techniques when elucidating protein structure and function. For example, chemical modification of leukotriene A4 hydrolase, 3-hydroxyisobutyrate dehydrogenase, and lactate dehydrogenase (1-3) have contributed significantly to the understanding of active-site mechanisms in these proteins and in elucidating the mechanisms of DNA binding in the Fd and Pf 1 gene 5 proteins (4–5).

Glycogen synthase kinase-3 (GSK-3) is required during metazoan development to mediate the effects of the extracellular signal wingless/Wnt-1 and hence is necessary for correct cell type specification. GSK-3 also regulates cell fate during Dictyostelium development, but in this case it appears to mediate the effects of extracellular cAMP. By direct measurement of GSK-3 kinase activity during Dictyostelium development, we find that there is a rise in activity at the initiation of multicellular development which can be induced by cAMP. The timing of the rise correlates with the requirement for the Dictyostelium homologue of GSK-3, GSKA, to specify cell fate. We show that loss of the cAMP receptor cAR3 almost completely abolishes the rise in kinase activity and causes a mis-specification of cell fate that is equivalent to that seen in a gskA- mutant. The phenotype of a cAR3(-) mutant however is less severe than loss of gskA and ultimately gives rise to an apparently wild-type fruiting body. These results indicate that in Dictyostelium extracellular cAMP acts via cAR3 to cause a rise in GSKA kinase activity which regulates cell type patterning during the initial stages of multicellularity.

We report the cloning of the skp1(+) gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1(+) gene is not essential. However, cells with deletions in skp1(+) are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1(+) specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (M(r) 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances in the H-1 NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-teminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the LH NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the T-m of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.

Extracellular cyclic AMP (cAMP) induces the formation of prespore cells in Dictyostelium but inhibits stalk cell formation. We have cloned gskA, which encodes the Dictyostelium homolog of glycogen synthase kinase 3 (GSK-3), and discovered that it is required for both cAMP effects. Disruption of gskA creates a mutant that aggregates but forms few spores and an abnormally high number of stalk cells. These stalk cells probably arise from an expanded prestalk B (pstB) cell population, which normally produces the basal disc of the fruiting body. In cultured mutant cells, cAMP neither inhibits pstB cell differentiation nor induces efficient prespore cell differentiation. We propose that cAMP acts through a common pathway that requires GSK-3 and determines the proportion of prespore and pstB cells.

The glycogen synthase kinase-3 (GSK-3) family of protein-serine kinases is implicated in the development and hormonal regulation of higher eukaryotes. GSK-3-related genes have been cloned and characterized in mammals (α and β forms), Drosophila melanogaster (shaggy/zeste-white3) and Saccharomyces cerevisiae (MCK1). Using the polymerase chain reaction and primers designed to hybridize to conserved catalytic domain sequences of this family, a genomic fragment was amplified from budding yeast DNA. Genomic clones encompassing the entire reading frame were subsequently isolated and sequenced. The protein encoded by this gene, termed ScGSK-3, displays high identity with members of the GSK-3 family, sharing several structural features including a regulatory Tyr residue. A phylogenetic analysis of the catalytic domains of these protein kinases suggests that ScGSK-3 represents the bona fide homologue of GSK-3 and the shaggy product, while the related MCK1 protein kinase is encoded by a paralogous gene which originated by a gene duplication event in the yeast lineage.

Glycogen synthase kinase‐3 (GSK‐3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste‐white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c‐Jun/AP‐1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK‐3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK‐3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK‐3 family and is equivalent to that required for activity by mitogen‐activated protein (MAP) kinases. However, unlike MAP kinases, GSK‐3 is highly phosphorylated on tyrosine and thus active in resting cells.

The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments. The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence. The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex. Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA. Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein. The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex.

Conference Article| November 01 1993 Roles of glycogen synthase kinase-3 in signal transduction James R. Woodgett; James R. Woodgett 1Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada Search for other works by this author on: This Site PubMed Google Scholar Simon E. Plyte; Simon E. Plyte 1Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada Search for other works by this author on: This Site PubMed Google Scholar Bernd J. Pulverer; Bernd J. Pulverer 1Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada Search for other works by this author on: This Site PubMed Google Scholar Jennifer A. Mitchell; Jennifer A. Mitchell 1Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada Search for other works by this author on: This Site PubMed Google Scholar Ken Hughes Ken Hughes 1Division of Cell and Molecular Biology, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (4): 905–907. https://doi.org/10.1042/bst0210905 Article history Received: July 26 1993 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation James R. Woodgett, Simon E. Plyte, Bernd J. Pulverer, Jennifer A. Mitchell, Ken Hughes; Roles of glycogen synthase kinase-3 in signal transduction. Biochem Soc Trans 1 November 1993; 21 (4): 905–907. doi: https://doi.org/10.1042/bst0210905 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: GSK-3, glycogen synthase kinase-3, MAP kinase, mitogen-activated protein kinase, PKC, protein kinase C This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

Study of GSK-3 had an inauspicious beginning rooted in intermediary metabolism. However, owing to the fortuitous convergence of several disparate areas of biology, the enzyme now offers unique opportunities for study of the control of a variety cellular processes. While at first sight a role in transcriptional regulation appears unlikely for a protein first identified as acting on glycogen synthase, it is even more surprising that the same protein should be functionally interchangeable with a fruit fly homeotic gene. Such understandable scepticism, however, is based on teleological bias. Glycogen synthase is a critical enzyme regulating glucose storage. The c-Jun oncoprotein may have the potential to transform cells but this does not excuse it from similar mechanisms of control to glycogen synthase. Likewise, homeotic genes play a crucial role in setting up the body plan of an embryo but must also be subject to control. The main difference is that when such control is lost, the result is rather graphic. It is, therefore, only to be expected that regulatory protein kinases will surface in superficially quite unrelated areas and that many of their targets will be 'housekeeping' proteins. Perhaps the most difficult aspect of protein phosphorylation research is the linking of physiological substrates with particular protein kinases, hence reconstructing pathways. No matter how compelling in vitro data appear, there must be demonstration that the protein is targeted by the specific protein kinase in cells, an extremely difficult process. Most progress in this respect has been made using genetic analysis in lower organisms, especially yeast. Here another problem arises: demonstration of biochemical linkages underlying genetic interactions which requires function to be ascribed to genes identified by a gross effect. The challenge is to co-ordinate these two approaches, a strategy currently being employed to further unravel the biological role of GSK-3.

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pfl. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/Å) indicates an axial subunit repeat of 5·1 Å, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43·3 Å) indicates an outer radius of 60 to 63 Å for the complex. The variation in Rc with contrast shows that regions of higher scattering are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0·105 Å−1 arising from the 60 Å helical pitch of the nucleoprotein complex. The structural parameters of the Pfl gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.

The 144 amino acid gene 5 protein of bacteriophage Pf1 binds tightly and cooperatively to single-stranded DNA during replication of the phage genome. It has been suggested that aromatic amino acid side chains are important for this interaction, probably through base stacking with the DNA. We have analysed the accessibility of tyrosine residues in the DNA - protein complex, and their importance to the DNA-binding activity of the protein, by chemical modification and protection experiments using tetranitromethane. Tyrosines 21, 30 and 55 are surface accessible in the free protein but are protected from modification in the complex with phage DNA. Moreover, modification of these residues in the free protein abolishes the ability to bind to DNA or oligonucleotides, as judged by fluorescence spectroscopy and gel retardation analysis. Modification of the protein also results in the formation of an intersubunit covalent cross-link between Tyr55 and Phe76, suggesting that Phe76 is located within the DNA-binding cleft of the protein. It is proposed that residues 17 - 34 of the Pf1 gene 5 protein form a beta-hairpin analogous to the 'DNA-binding wing' of the fd and Ike gene 5 proteins. We suggest the existence of a single-stranded DNA binding motif, in which Tyr30 of the Pf1 protein is equivalent to the functionally important Tyr26 of the fd gene 5 protein.

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.

Conference Article| February 01 1989 Sedimentation analysis of the Pf1 gene 5 protein P. J. MORGAN; P. J. MORGAN *Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington LE12 5RD, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. E. HARDING; S. E. HARDING *Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington LE12 5RD, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. E. PLYTE; S. E. PLYTE †Biophysics Laboratory, Portsmouth Polytechnic, Portsmouth, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. G. KNEALE G. G. KNEALE †Biophysics Laboratory, Portsmouth Polytechnic, Portsmouth, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1989) 17 (1): 234–235. https://doi.org/10.1042/bst0170234 Article history Received: July 23 1988 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation P. J. MORGAN, S. E. HARDING, S. E. PLYTE, G. G. KNEALE; Sedimentation analysis of the Pf1 gene 5 protein. Biochem Soc Trans 1 February 1989; 17 (1): 234–235. doi: https://doi.org/10.1042/bst0170234 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: M0LW, whole-cell average relative molecular mass, MLW, point weight average relative molecular mass This content is only available as a PDF. © 1989 Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

Limited proteolysis is a useful structural probe for investigating the globular nature of proteins by preferentially digesting the more accessible regions often found between domains. Generally, proteases require a small region of polypeptide chain possessing conformational flexibility for accommodation in the active site (). The regions of a protein possessing conformational flexibility are often found between tightly folded domains and are therefore preferential sites for proteolysis. In practice, limited proteolysis is achieved by dilution of the enzyme sufficiently so that it will only digest the most accessible regions, leaving the domains intact. Digestion of protein-nucleic acid complexes is often advantageous in that the DNA may provide steric protection of the DNA binding domain not afforded by the free protein. The generation of domains by limited proteolysis relies directly on the tertiary structure of the protein under investigation and provides much firmer evidence for their existence than that provided by sequence homology.