Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.

Type I interferons (IFNs) 1 bind to a multimeric receptor (type I IFN-R, IFN␣R, or IFN␣␤R) composed of at least two subunits designated as ␣ (1-5), and ␤ (6, 7). The IFN␣/␤R cDNA (8) corresponds to the short form of the ␤ subunit (␤ S ), whereas cDNAs for the long form (␤ L ) have been recently isolated by two different groups (9, 10). Transcripts and mature proteins for both ␤ S and ␤ L are found in most cell lines, although there is a large excess of ␤ L protein when compared with ␤ S .2 Lutfalla et al. (10) have also shown that the short and long forms of the ␤ subunit are generated by alternative splicing of the same gene. Interestingly, the type I IFN-binding proteins are not limited to the cellular components described above, because we and others have found a vaccinia virusencoded protein capable of binding type I IFNs as well as blocking type I IFN signaling (11,12).Coexpression of the ␣ subunit with either ␤ L or ␤ S in mouse L-929 cells reconstitutes the high affinity receptor, whereas either form of the ␤ chain expressed independently binds type I IFNs with low affinity. Additional studies using these transfectants show that the IFN␣ response can be reconstituted in mouse cells only when the ␣ chain is coexpressed with the ␤ L subunit rather than ␤ S , indicating that both the ␣ and ␤ L subunits are indispensable for IFN␣ signaling (9). Similarly, only the long form of the ␤ subunit is able to complement the mutant cell line U5A that is defective in the IFN␣ pathway (10).IFNs, cytokines, and growth factors activate the Jak-Stat pathway; more specifically, type I IFNs activate Jak1, Tyk2, Stat1, Stat2, and Stat3 (for recent reviews, see Refs. 13 and 14). Ligand binding to the cytokine receptor subunits activates tyrosine kinases of the Jak family that are constitutively associated with different cytokine receptors. It has been demonstrated that Jak2 binds to the Box 1 motif present in the membrane proximal region of the erythropoietin, prolactin, growth hormone, and granulocyte macrophage colony-stimulating factor receptors (15-21). In the case of the ␣ subunit of the type I IFN-R, there is no conserved Box 1 motif, and Tyk2 interacts with a distinct region different from that of Box 1 (22). Furthermore, in those cytokine receptors that activate Jak1, the region that interacts with this kinase has not been clearly defined. The presence of a Box 1 motif in the membrane proximal region of the ␤ L subunit of the type I IFN-R suggested a possible role for this motif in the binding and/or activation of Jak1 by this receptor subunit. We therefore sought to determine the role of the Box 1 and other domains of the ␤ L chain in type I IFN signaling using two strategies: (a) we stably cotransfected mouse L-929 cells with the wild-type ␣ subunit and various truncated forms of the ␤ L chain to study in vivo signaling processes; and (b) we produced GST fusion proteins with deletions of the ␤ L chain to demonstrate interactions in vitro. Our data indicate that a minimal region of the ␤ L subunit cytoplasmic domain,...

Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (␣␤␥)␦ 4 . The ␥ subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the ␣␤␥␦ protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily ␣␤ dimers, plus smaller amounts of ␤␥␥ and ␣␥␥ trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o-and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity crosslinker in binding to these sites prior to forming crosslinked conjugates. The largest effect on cross-linking in progressing from o-to p-PDM was on the ␣␥␥ trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of ␣␥␥ formed, small increases in ␤␥␥, and little change in ␣␤. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic ␥ subunit with the regulatory ␣ and ␤ subunits upon activation of PbK.Through allosteric and covalent modification sites on its three regulatory subunits, phosphorylase b kinase (PbK) 1 integrates neural, hormonal, and metabolic signals to modulate glycogenolytic flux in skeletal muscle (for review, see Refs. 1 and 2). Although the ␣, ␤, and ␦ (calmodulin) regulatory subunits clearly control the activity of the catalytic ␥ subunit, little is known concerning the mechanisms through which they exert this control, including the extent to which their regulatory influence on ␥ is direct versus indirect; this is especially the case for the larger regulatory subunits, ␣ and ␤. Phosphorylation (3) or proteolysis (4) of ␣ causes increased activity of ␥ within the (␣␤␥␦) 4 holoenzyme, but evidence for a direct ␣-␥ interaction that is altered by this activation has not been observed. Likewise, multiple means of activating the holoenzyme cause common conformational changes in the ␤ subunit (5-7); but again, no evidence for alteration of a direct ␤-␥ interaction has been observed. In two previous studies, cross-linking was used successfully to detect changes in the cross-linking patterns of both the ␣ (8) and ␤ (5) subunits upon activation of the holoenzyme; but with the cross-linkers used, the observed changes involved only a second ␣ or ␤ subunit to form homod...

Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-GlcNAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t1 ⁄2 < 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED 50 ‫؍‬ 4 mM). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mM) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min (ϳ400 nmol/g) and continued to rise over 2 h (t1 ⁄2 ϳ 20 min) before reaching a plateau at >1,400 nmol/g (ED 50 ‫؍‬ 900 M). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDPGlcNAc levels (t1 ⁄2 ϳ 14 -18 min), but greater levels were obtained with GlcN (4 -5-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 M, ED 50 ‫؍‬ 80 M) could markedly elevate UDPGlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.

Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex.

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist-or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC 50 (Ca 2؉ ) The nitric-oxide synthases catalyze formation of NO and L-citrulline from L-arginine and O 2 , with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS) 2 in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130 -160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca 2ϩ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca 2ϩ ) 4 -CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca 2ϩ

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca 2؉ ions, of the (␣␤␥␦) 4 phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory ␣ and catalytic ␥ subunits. The ␥ subunit is also known to interact with the ␦ subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca 2؉ ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the ␥ (residues 343-386) and ␣ (residues 1060 -1237) subunits interact. The association of this region of ␣ with ␥ was corroborated by the isolation of a crosslinked fragment of ␣ containing residues 1015-1237 from an ␣؊␥ dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of ␥ that we found to interact with ␣ has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156 -17163), we tested the influence of Ca 2؉ on the conformation of the ␣ subunit and found that the region of ␣ that interacts with ␥ was, in fact, perturbed by Ca 2؉ . The results herein support the existence of a Ca 2؉ -sensitive communication network among the ␦, ␥, and ␣ subunits, with the regulatory domain of ␥ being the primary mediator. The similarity of such a Ca 2؉ -dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the ␥ subunit of PhK.Phosphorylase kinase (PhK) 1 , a Ca 2ϩ -dependent enzyme involved in the regulation of glycogenolysis, is among the largest and most complex enzymes known. Structurally, PhK is composed of four copies each of four different subunits, (␣␤␥␦) 4 and has a mass of 1.3 ϫ 10 6 Da (for reviews see Refs. 1-3). Of the four subunits, ␥ is catalytic, whereas the remaining three are regulatory: ␣ and ␤ exert quaternary constraint on the activity of ␥, and ␦ is an intrinsic molecule of calmodulin (CaM). To fully understand how PhK integrates diverse physiological signals to regulate glycogenolytic flux in skeletal muscle, it is first essential to understand how intrasubunit and intersubunit interactions within the hexadecameric holoenzyme change in response to effector ligands, and in so doing, control its catalytic activity. Despite the increased availability of structural information regarding PhK, interactions associated with activation and involving specific regions of individual subunits, in particular the ␣ and ␤ subunits, have largely remained uncharacterized. In this study, we have focused on delineating interacting regions between the large ␣ and catalytic ␥ subunits to advance our understanding of how structural perturbations correlate with activation of this complex holoenzyme.By using chemical cross-linkers as s...

The signaling specificity for cytokines that have common receptor subunits is achieved by the presence of additional cytokine-specific receptor components. In the type I interferon (IFN) family, all 14 subtypes of IFN␣, IFN␤, and IFN bind to the same ␣ and ␤ L subunits of the type I IFN-R, yet differences in signaling and biological effects exist among them. Our data demonstrate that IFN␣2 and IFN␤ utilize different regions of the ␤ L subunit for signaling. Thus, in contrast to other cytokine systems, signal diversity in the type I IFN system can be accomplished within the same receptor complex by utilizing different regions of the same receptor subunits.Ligand binding to a receptor induces oligomerization of receptor subunits, which results in activation of various signaling pathways. Specificity in ligand receptor systems is achieved at the extracellular level by the specific interaction of a ligand with its distinct receptor components and at the intracellular level by the interaction of the cytoplasmic domain of the receptor subunits with a distinct set of signal transducing proteins. Many cytokine systems share receptor subunits (1-3), and in these situations it is commonly accepted that specificity is determined by the existence of additional ligand-specific receptor chains. For instance, the IL2R 1 has a binding subunit (␣ chain) and two signaling chains designated as ␤ and ␥c. The IL2R␥c chain is also common to the IL4, IL7, and IL9 receptors and functions in association with specific receptor subunits for each of these cytokines (i.e. IL4R␣, IL7R␣, and IL9R␣ chains). Therefore, these cytokines have the ability to produce both redundant and distinct biological effects (1-3).The type I interferon (IFN) family includes 14 subtypes of IFN␣, as well as one IFN␤ and IFN, all of which bind to the same cell surface receptor designated as IFN␣R, IFN␣␤R, or type I IFN-R (4). The type I IFN-R is composed of at least two subunits: the ␣ chain or IFNAR1 (5-9) and the ␤ subunit or IFNAR2, which has short (␤ S ) and long (␤ L ) forms (10 -14). Although expression of the ␣ chain with either ␤ L or ␤ S produces high affinity receptors in murine L-929 cells, only coexpression of ␣ and ␤ L allows activation of the Jak-Stat pathway and induction of an antiviral state in response to stimulation by both huIFN␣2 and huIFN␤ (13,14). Interestingly, although both of these human IFNs bind to the same receptor and activate the same components of the Jak-Stat pathway, there are some signaling and biological differences. IFN␤ signaling has two distinctive features: (i) induction of a very strong association of the ␣ and ␤ L subunits of the type I IFN-R (15) and (ii) transcriptional activation of the ␤-R1 gene (16). These signaling differences could be responsible for the disparity in biological effects among the members of the IFN␣ family. For example, IFN␤ is more effective than other type I IFNs in the treatment of multiple sclerosis (17, 18). However, unlike other cytokines, the differences in signaling and biological activities ...