Carol J. Fiol

Binding of GSK3β to the APC-β-Catenin Complex and Regulation of Complex Assembly

The adenomatous polyposis coli gene (APC) is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule β-catenin, which is a mammalian homolog of ARMADILLO, a component of the WINGLESS signaling pathway in Drosophila development. Here it is shown that when β-catenin is present in excess, APC binds to another component of the WINGLESS pathway, glycogen synthase kinase 3β (GSK3β), a mammalian homolog of Drosophila ZESTE WHITE 3. APC was a good substrate for GSK3β in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of β-catenin to this region was dependent on phosphorylation by GSK3β.

Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1.

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.

Identification of phosphorylation sites in peptides using a gas-phase sequencer☆

A simple procedure is described for determining the location of phosphorylation sites in phosphopeptides. The method employs measurement of 32P-labeled inorganic phosphate release during Edman degradation cycles using a gas-phase sequencer. The procedure is based on extracting peptides and inorganic phosphate from portions of the sample filter at strategic cycles in the sequence analysis followed by determination of the relative amounts of phosphate and phosphopeptide. One advantage of this technique is the very high recovery of the phosphate associated with the peptide, 80-97% in this study. In the course of this work, it was also found that phosphoserine residues themselves caused reduced efficiency of the Edman degradation as compared with unesterified serine residues. The present procedure has the merit of being simple and easy to apply.

Glycogen metabolism and signal transduction in mammals and yeast

Mammalian glycogen synthase, with its complex multisite phosphorylation mechanisms, continues to provide interesting and novel examples of the regulation of protein function. The mammalian enzyme is phosphorylated in a hierarchal manner such that modification of certain sites requires the prior phosphorylation of other sites. Yeast contains two glycogen synthases that have extensive similarities to their mammalian counterpart but the greatest divergence in amino acid sequence is seen precisely in the regions likely to be involved in covalent control. We hope that examination of the control of the yeast glycogen synthase will be as informative as study of the mammalian enzymes, whether by revealing important parallels with the mammalian system or by uncovering major differences in mechanism.