Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK, JNK (SAPK), ERK5 (BMK), and p38. Here we characterize a new MAP kinase, p38␤. p38␤ is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site, which is required for its kinase activity. Like p38, p38␤ is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38␤ and p38 revealed differences, most notably in the preferred activation of p38␤ by MAP kinase kinase 6 (MKK6), whereas p38 was activated nearly equally by MKK3, MKK4, and MKK6. Moreover, in vitro and in vivo experiments showed a strong substrate preference by p38␤ for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38␤ was ϳ20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related, p38␤ and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets.

Genetic studies of molecules that negatively regulate G-coupled receptor functions have led to the identification of a large gene family with an evolutionarily conserved domain, termed the RGS domain. It is now understood that RGS proteins serve as GTPase-activating proteins for subfamilies of the heterotrimeric G-proteins. We have isolated from mouse pituitary a fulllength cDNA clone encoding a novel member of the RGS protein family, termed RGS16, as well as the full-length cDNA of mRGS5 and mRGS2. Tissue distribution analysis shows that the novel RGS16 is predominantly expressed in liver and pituitary, and that RGS5 is preferentially expressed in heart and skeletal muscle. In contrast, RGS2 is widely expressed. Genetic analysis using the pheromone response halo assay and FUS1 gene induction assay show that overexpression of the RGS16 gene dramatically inhibits yeast response to ␣-factor, whereas neither RGS2 nor RGS5 has any discernible effect on pheromone sensitivity, pointing to a possible functional diversity among RGS proteins. In vitro binding assays reveal that RGS5 and RGS16 bind to G␣ i and G␣ o subunits of heterotrimeric G-proteins, but not to G␣ s . Based on mutational analysis of the conserved residues in the RGS domain, we suggest that the G-protein binding and GTPase-activating protein activity may involve distinct functional structures of the RGS proteins, indicating that RGS proteins may exert a dual function in the attenuation of signaling via G-coupled receptors.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for G i , G q , and G 12 ␣-subunits of heterotrimeric G proteins. Although it is known that membrane association is critical for the biological activities of many RGS proteins, the mechanism underlying this requirement remains unclear. We reported recently that the NH 2 terminus of RGS16 is required for its function in vivo. In this study, we show that RGS16 lacking the NH 2 terminus is no longer localized to the plasma membrane as is the wild type protein, suggesting that membrane association is important for biological function. The region of amino acids 7-32 is sufficient to confer the membrane-targeting activity, of which amino acids 12-30 are predicted to adopt an amphipathic ␣-helix. Site-directed mutagenesis experiments showed that the hydrophobic residues of the nonpolar face of the helix and the strips of positively charged side chains positioned along the polar/nonpolar interface of the helix are crucial for membrane association. Subcellular fractionation by differential centrifugation followed by conditions that distinguish peripheral membrane proteins from integral ones indicate that RGS16 is a peripheral membrane protein. We show further that RGS16 membrane association does not require palmitoylation. Our results, together with other recent findings, have defined a unique membrane association domain with amphipathic features. We believe that these structural features and the mechanism of membrane association of RGS16 are likely to apply to the homologous domains in RGS4 and RGS5.Regulators of G protein signaling (RGS 1 proteins) have emerged as major modulators of diverse aspects of biological activities (1-4). It has been well established that RGS proteins act as GTPase-activating factors for many of the heterotrimeric G protein ␣-subunits (5-7). The core RGS domain is responsible for GTPase-activating protein activity, although it is not sufficient for biological function in vivo (8 -12).Many RGS proteins have been shown to be membrane-associated. GAIP behaves as an integral membrane protein as judged by its resistance to the stripping effect of Na 2 CO 3 treatment (13). Plasma membrane association has also been shown to be required for RGS4 function (12). RGSZ1, a G z -selective RGS protein in the brain, is also tightly membrane-bound and requires membrane association for its GTPase-activating protein activity toward G z (14, 15). However, it is unclear how they are targeted to the membrane. RET-RGS1, another potentially membrane-bound RGS member, contains a putative transmembrane domain and multiple cysteine residues for palmitoylation (9). GAIP and RGS4 also possess cysteine string motifs in their NH 2 terminus and have been shown to be palmitoylated (12, 13). Although palmitoylated GAIP is found only in pellet fractions, the functional significance of palmitoylation has yet to be established. Surprisingly, palmitoylation of RGS4 is shown not to be required for...

The regulators of G-protein signaling (RGS) family members contain a conserved region, the RGS domain, and are GTPase-activating proteins for many members of G-protein K Ksubunits. We report here that the core domain of RGS16 is sufficient for in vitro biochemical functions as assayed by its Gprotein binding affinity and its ability to stimulate GTP hydrolysis by GK K o protein. RGS16 also requires, in addition to the RGS domain, the divergent N-terminus for its biological function in the attenuation of pheromone signaling in yeast, whereas its C-terminus region is dispensable. Together with other evidence, these data support the notion that RGS proteins interact with other cellular factors and may serve to link specific G-proteins to different downstream effectors in G-proteinmediated signaling pathways.z 1998 Federation of European Biochemical Societies.

Regulators of G-protein signaling (RGS) are GTPase-activating proteins (GAP) for activated GK K subunits. We found that mouse RGS16, when expressed in HEK293T cells, is phosphorylated constitutively at serine 194 based on in vivo orthophosphate labeling experiments, while serine 53 is phosphorylated in a ligand-dependent manner upon stimulation by epinephrine in cells expressing the K K2A adrenergic receptor. Phosphorylation on both sites impairs its GAP activity and subsequent attenuation on heterotrimeric G-protein-stimulated extracellular signal-regulated protein kinase activity. This is the first report of RGS functional downregulation by phosphorylation via a G-protein-coupled receptor. ß

Nek2 has been implicated in centrosome disjunction at the onset of mitosis to promote bipolar spindle formation, and hyperactivation of Nek2 leads to the premature centrosome separation. Its activity, therefore, needs to be strictly regulated. In this study, we report that Cep85, an uncharacterized centrosomal protein, acts as a binding partner of Nek2A. It colocalizes with isoform A of Nek2 (Nek2A) at centrosomes and forms a granule meshwork enveloping the proximal ends of centrioles. Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction. By contrast, depletion of Cep85 results in the precocious centrosome separation. We also define the Nek2A binding and centrosome localization domains within Cep85. Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro, both domains are indispensable for full suppression of centrosome disjunction in cells. Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells.

The kinetics for complete iron release showing biphasic behavior from pig spleen ferritin-Fe (PSFF) was measured by spectrophotometry. The native core within the PSFF shell consisted of 1682 hydroxide Fe3+ and 13 phosphate molecules. Inhibition kinetics for complete iron release was measure by differential spectrophotometry in the presence of phosphate; the process was clearly divided into two phases involving a first-order reaction at an increasing rate of 46.5 Fe3+/PSFF/min on the surface of the iron core and a zero-order reaction at a decreasing rate of 6.67 Fe3+/PSFF/min inside the core. The kinetic equation [C(PSFF-Fe3+)max - C(PSFF-Fe3+)t](1/2) = Tmax - Tt gives the transition time between the two rates and represents the complex kinetic characteristics. The rate was directly accelerated twofold by a mixed reducer of dithionite and ascorbic acid. These results suggest that the channel of the PSFF shell may carry out multiple functions for iron metabolism and storage and that the phosphate strongly affects the rate of iron release.