Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein.

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.     

Interaction of SNF1 protein kinase with its activating kinase Sak1

The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.     

Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.     

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.     

A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.

The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.     

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex

BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.     

Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase.

The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.     

An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.     

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.     

Biochemical and functional studies on the regulation of the Saccharomyces cerevisiae AMPK homolog SNF1

AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1, Snf1 in yeast, contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID), and a region that mediates interactions with the two regulatory (β and γ) subunits. Previous studies suggested that Snf1 contains an additional segment, a regulatory sequence (RS, corresponding to residues 392-518), which may also have an important role in regulating the activity of the enzyme. The crystal structure of the heterotrimer core of Saccharomyces cerevisiae SNF1 showed interactions between a part of the RS (residues 460-498) and the γ subunit Snf4. Here we report biochemical and functional studies on the regulation of SNF1 by the RS. GST pulldown experiments demonstrate strong and direct interactions between residues 450-500 of the RS and the heterotrimer core, and single-site mutations in the RS-Snf4 interface can greatly reduce these interactions in vitro. On the other hand, functional studies appear to show only small effects of the RS-Snf4 interactions on the activity of SNF1 in vivo. This suggests that residues 450-500 may be constitutively associated with Snf4, and the remaining segments of the RS, as well as the AID, may be involved in regulating SNF1 activity.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

Ptc1 Protein Phosphatase 2C Contributes to Glucose Regulation of SNF1/AMP-activated Protein Kinase (AMPK) in Saccharomyces cerevisiae

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.     

Cloning and characterization of PHIP, a novel insulin receptor substrate-1 pleckstrin homology domain interacting protein

Insulin receptor substrate-1 (IRS-1) protein is a major substrate of the insulin receptor tyrosine kinase and is essential for transducing many of the biological effects of insulin including mitogenesis, gene expression, and glucose transport. The N terminus of IRS-1 contains a pleckstrin homology (PH) domain that is critical for recognition and subsequent phosphorylation of IRS-1 by the activated insulin receptor. Here we report the isolation of a novel protein, PHIP (PH-interacting protein), which selectively binds to the PH domain of IRS-1 in vitro and stably associates with IRS-1 in vivo. Importantly, mutants of the IRS-1 PH domain that disrupt the PH fold fail to bind to PHIP. Anti-phosphotyrosine immunoblots of PHIP revealed no discernible insulin receptor-regulated phosphorylation, suggesting that PHIP is not itself a substrate of the insulin receptor. In contrast to full-length PHIP, overexpression of the PH-binding region of PHIP has a pronounced inhibitory effect on insulin-induced IRS-1 tyrosine phosphorylation levels. Furthermore, expression of this dominant-negative PHIP mutant leads to a marked attenuation of insulin-stimulated mitogen-activated protein kinase activity. We conclude that PHIP represents a novel protein ligand of the IRS-1 PH domain that may serve to link IRS-1 to the insulin receptor.     

Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation.

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

P53与TRF1、TRF2的体外结合及P53结合功能区的分析

通过分析端粒结合蛋白TRF1、TRF2与P5 3的体外结合 ,探讨P5 3 端粒途径调节细胞生命活动的分子机制 .GST和 4种人P5 3 GST融合蛋白经大肠杆菌表达、谷胱甘肽 SepharoseTM4B纯化后 ,进行SDS PAGE和考马斯亮篮染色 .人P5 3包括野生型 (1～ 393)、C端缺失体P5 3N5 (2～ 2 93)、N端缺失体P5 32C(95～ 393)、单个氨基酸突变体P5 3R175H(175R→H) .各纯化蛋白的分子量与预计的完全一致 ,且纯化率达 90 %以上 .将纯化的GST和P5 3 GST融合蛋白与人乳腺癌细胞MCF 7细胞蛋白进行体外结合反应 ,Western印迹检测反应物中P5 3和TRF1、TRF2的结合 .野生型P5 3和P5 3 R175H均能沉淀MCF 7中的TRF1、TRF2 ,且结合力相似 ,而单独的GST则无沉淀TRF1、TRF2的作用 .与野生型P5 3和P5 3R175H相比 ,P5 32C与TRF1、TRF2的结合力明显增加 ,P5 3N5与TRF1、TRF2的结合力大大减弱 .表明P5 3和TRF1、TRF2可以进行直接而特异的体外结合 ,且它们的结合为P5 3C端 (2 93～ 393)结构域依赖性 .P5 3和TRF1、TRF2这种结构域依赖性的结合可能与端粒动态变化所诱导的细胞活动有关 .     

Structure of the frequency‐interacting RNA helicase: a protein interaction hub for the circadian clock

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ‐interacting RNA Helicase (FRH) rhythmically represses gene expression by the white‐collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ‐interacting region at the FRH N‐terminus has variable structure in the absence of FRQ. A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ. A V142G substitution near the N‐terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N‐terminus and KOW module.