Although the general pathways of glycogen synthesis and glycogenolysis are identical in all tissues, the enzymes involved are uniquely adapted to the specific role of glycogen in different cell types. In liver, where glycogen is stored as a reserve of glucose for extrahepatic tissues, the glycogen-metabolizing enzymes have properties that enable the liver to act as a sensor of blood glucose and to store or mobilize glycogen according to the peripheral needs. The prime effector of hepatic glycogen deposition is glucose, which blocks glycogenolysis and promotes glycogen synthesis in various ways. Other glycogenic stimuli for the liver are insulin, glucocorticoids, parasympathetic (vagus) nerve impulses and gluconeogenic precursors such as fructose and amino acids. The phosphorolysis of glycogen is mainly mediated by glucagon and by the orthosympathetic neurotransmitters noradrenaline and ATP. Many glycogenolytic stimuli, e.g. adenosine, nucleotides and NO, also act indirectly, via secretion of eicosanoids from non-parenchymal cells. Effectors often initiate glycogenolysis cooperatively through different mechanisms.

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Specific vasopressin binding to rat hepatocytes and rat liver membranes was measured using biologically active (3H)-Tyr2-Lys8-vasopressin (8.5 Ci/mM). In both systems, vasopressin binding was found to be time-dependent, reversible, and saturable. The kinetic parameters for vasopressin binding were: apparent dissociation constants (Kd): 4.9 nM and 15 nM; maximal binding capacities: 0.83 pmoles/mg protein and 2.10(5) sites/Cell for purified membranes and intact cells respectively. The relative affinities of 19 vasopressin structural analogues were deduced from competition experiments and compared to the previously determined glycogenolytic (or antiglycogenolytic) potencies of these analogues. For both agonists and antagonists, a highly significant correlation was demonstrated between pKd and pKa (or pKi) values, suggesting that the detected binding sites are the physiological receptors involved in the glycogenolytic action of vasopressin on the rat liver. The affinity of antagonists for binding to these receptors is the same for both membranes and cells. In contrast, agonists which bind to vasopressin receptor sites have a higher affinity for purified membranes than for intact cells (Kd membranes/Kd cells = 8 +/- 1). GTP (0.1mM) reduced the affinity of agonists but not of antagonists for binding to membranes and abolished the differences between Kd values for binding to hepatocytes and membranes.

NIPP1 is a ubiquitous nuclear protein that is required for spliceosome assembly. We report here that the phosphothreonine-binding Forkhead-associated domain of NIPP1 interacts with the cell cycle-regulated protein Ser/Thr kinase MELK (maternal embryonic leucine zipper kinase). The NIPP1-MELK interaction was critically dependent on the phosphorylaton of Thr-478 of MELK and was increased in lysates from mitotically arrested cells. Recombinant MELK was a potent inhibitor of an early step of spliceosome assembly in nuclear extracts. This splicing defect was also seen with a kinase-dead mutant but was absent after mutation (T478A) of the NIPP1 binding site of MELK, indicating a mediatory role for NIPP1. Our data suggest that MELK has a role in the cell cycle-regulated control of pre-mRNA splicing.The nuclear protein NIPP1 1 (39 kDa) was originally discovered as a potent and specific inhibitor of protein Ser/Thr phosphatase-1 (PP1), hence its name, nuclear inhibitor of PP1 (1-7).More recently, we have demonstrated that NIPP1 is also implicated in transcription as well as in pre-mRNA splicing by mechanisms that do not involve PP1 (8, 9). In transient transfection experiments, NIPP1 acted as a transcriptional repressor, which may be accounted for by the binding of the central and C-terminal domains of NIPP1 to the Polycomb protein, EED (embryonic ectoderm development) (9). The latter promotes transcriptional repression by the recruitment of a histone methyltransferase and histone deacetylases. NIPP1 also appears to be required for the assembly of spliceosomes, the protein-RNA complexes that catalyze pre-mRNA splicing (8). The spliceosomal function of NIPP1 requires its C-terminal domain as well as its N-terminal Forkhead-associated (FHA) domain, an established phosphothreonine-binding module. The FHA domain of NIPP1 mediates targeting to both the spliceosomes and the nuclear storage sites for splicing factors, known as "speckles" (8, 10). The targeting function of the FHA domain of NIPP1 is likely explained by its ability to bind to phosphorylated forms of the essential splicing factors CDC5L (11) and SAP155 (12).Here we show that the protein kinase MELK, which is structurally related to the AMP-activated protein kinases, also interacts in a phosphorylation-dependent manner with the FHA domain of NIPP1 and that this interaction is increased during mitosis. Furthermore, we demonstrate that recombinant MELK blocks spliceosome assembly by a mechanism that involves NIPP1. Our data suggest a novel link between pre-mRNA processing and cell cycle progression. EXPERIMENTAL PROCEDURESYeast , cloned into the pEG202 vector in-frame with the LexA DNA-binding domain, was used as bait for the screening of a HeLa cell cDNA library (11). In this library, the cDNAs are subcloned behind a galactose-inducible promoter in the pJG4 -5 vector in-frame with the B42 activation domain. Interacting proteins were identified by growth of the yeast strain EGY188 in a Ϫleucine/ϩ galactose medium. The use of a plasmid-borne LacZ reporter gene (pSH18 ...

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1␥ 1 does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell ؊/؊ mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.Protein Ser/Thr phosphatase-1 (PP1) 1 is expressed in all eukaryotic cells and controls numerous cellular processes including metabolism, cell division, apoptosis, and protein synthesis (1-5). PP1 does not exist freely in the cell but is associated with a large variety of polypeptides that determine when and where the phosphatase acts. Currently, ϳ70 mammalian genes are already known to encode interactors of PP1 (4, 5). Some of these function as targeting subunits and bring PP1 in close proximity to its substrates. Others (also) modulate the activity and substrate specificity of PP1 or are themselves substrates for associated PP1. The available information suggests that proteins interact with PP1 via multiple short sequence motifs. These PP1-binding motifs can be shared among PP1 interactors, accounting for the ability of PP1 to form stable complexes with a large number of structurally unrelated proteins. The best characterized and most common PP1-binding motif conforms to the consensus sequence RKX 0 -1 VI{P}FW in which X denotes any residue and {P} any residue except Pro; it is often referred to as the RVXF motif (6). The RVXF motif binds to a hydrophobic channel near the C terminus of PP1 (7). The binding of the RVXF motif not only has a PP1 anchoring function but also promotes the interaction of secondary, lower affinity binding sites, often resulting in an altered activity and/or substrate specificity of PP1 (1).Mammalian genomes contain three genes that together encode four isoforms of PP1, namely PP1␣, PP1␤/␦, and the splice variants PP1␥ 1 and PP1␥ 2 (1, 5). These isoforms are ϳ90% identical at the protein level and differ mainly in their extremities. With the exception of PP1␥ 2 , which is only expressed in the testis and the brain, the mammalian PP1 isoforms are ubiquitously expressed. PP1␣, PP1␤/␦, and PP1␥ 1 show an overlapping but distinct subcellular localization (8). Within the nucleus PP1␣ is mainly associated with the nuclear matrix, PP1␤/␦ is enriched in the non-nucleolar chromatin fraction, and PP1␥ 1 is predominantly targeted to the nucleo...