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Protein kinases are organized in hierarchical networks that are assembled and regulated by scaffold proteins. Here, we identify the evolutionary conserved WD40-repeat protein Han11 as an interactor of the kinase homeodomain-interacting protein kinase 2 (HIPK2). In vitro experiments showed the direct binding of Han11 to HIPK2, but also to the kinases DYRK1a, DYRK1b and mitogen-activated protein kinase kinase kinase 1 (MEKK1). Han11 was required to allow coupling of MEKK1 to DYRK1 and HIPK2. Knockdown experiments in Caenorhabditis elegans showed the relevance of the Han11 orthologs Swan-1 and Swan-2 for the osmotic stress response. Downregulation of Han11 in human cells lowered the threshold and amplitude of HIPK2- and MEKK1-triggered signalling events and changed the kinetics of kinase induction. Han11 knockdown changed the amplitude and time dependence of HIPK2-driven transcription in response to DNA damage and also interfered with MEKK1-triggered gene expression and stress signalling. Impaired signal transmission also occurred upon interference with stoichiometrically assembled signalling complexes by Han11 overexpression. Collectively, these experiments identify Han11 as a novel scaffold protein regulating kinase signalling by HIPK2 and MEKK1. [ABSTRACT FROM AUTHOR]

The discovery of a second human gene, GYG2, encoding a liverspecific isoform of glycogenin, the self-glucosylating initiator of glycogen biosynthesis, raised the possibility for differential controls of this protein in liver and muscle. The new protein, glycogenin-2, had several properties similar biochemically to the muscle isoform, glycogenin-1, but unlike glycogenin-1, stable expression in fibroblasts led to a significant overaccumulation of glycogen. Ensuing attempts to generate reagents suitable for use with rodents, to examine the physiological regulation of glycogenin-2 by nutritional and hormonal factors, have been unsuccessful. Proof of a negative is difficult but the weight of the evidence is beginning to mitigate against the existence of a second glycogenin gene in rodents leading us to hypothesize that the presence of the GYG2 gene is limited to primates. [ABSTRACT FROM AUTHOR]

Glycogenin, a self-glucosylating protein involved in the initiation of glycogen biosynthesis, varies in intracellular concentration from barely detectable in liver to a high level in muscle. The effect of increasing the glycogenin level on glycogen synthesis was studied in rat 1 fibroblasts stably overexpressing rabbit muscle glycogenin. In the presence of glucose, all of the expressed glycogenin was attached to polysaccharide and the free protein could only be detected by western blot analysis after incubation of cells in a glucose-depleted medium or treatment of the cell extract with α-amylase. In control cells, increased extracellular glucose concentrations promoted translocation of glycogen synthase from the soluble to the pellet fraction with an increase in the associated glycogen. Overexpression of glycogenin did not affect total intracellular glycogen and glycogen synthase levels at any concentration of glucose but significantly reduced glucose-induced accumulation of insoluble glycogen and translocation of glycogen synthase. Immunofluorescence analysis revealed a diffuse cytoplasmic distribution of glycogenin expressed in rat 1 cells. In rat 1 cells incubated with glucose, discrete deposits of glycogen were detected by staining with HIO4/Schiff but this was eliminated by overexpressing glycogenin. Analysis of [14C]glucose-or [35S]methionine-labeled extracts from glycogenin-expressing cells by continuous polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis revealed a continuum of glycogenin-containing species from low molecular mass to sizes significantly greater than 400 kDa. We conclude that (a) overexpression of glycogenin does not enhance glycogen synthesis but causes production of more, smaller, glycogen molecules with a concomitant change in their intracellular localization; (b) glycogenin and elevated glucose have opposing effects on the distribution of glycogen and glycogen synthase in rat 1 cells; and (c) the biogenesis of glycogen in rat I cells occurs without the accumulation of any major intermediate form. [ABSTRACT FROM AUTHOR]