Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2.alpha. kinase

Matts, Robert L.; Hurst, Robin; Xu, Zuoyu

doi:10.1021/bi00080a001

Cited by 55 publications

(46 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, mutations in DnaJ or DnaK (Hsp70) in E. coli reduce the phosphorylation of threonyl-tRNA synthetase and glutamyl-tRNA synthetase (14,27) by mechanisms that have not yet been clarified. In mammalian cells, Hsp70 was found to associate with the heme-dependent serine/threonine kinase and to influence its activity during heat shock in response to the accumulation of abnormal proteins (17)(18)(19). In this case, the chaperones affect the kinase rather than the polypeptide substrate.…”

Section: Discussionmentioning

confidence: 99%

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Yaglom

Goldberg

Finley

et al. 1996

Molecular and Cellular Biology

View full text Add to dashboard Cite

The G 1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34 CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34 CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34 CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34 CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.In Escherichia coli, mitochondria, and the cytosol of eukaryotic cells, members of DnaJ family of molecular chaperones function together with Hsp70s in protein folding and translocation across membranes (12). Recent studies have demonstrated that the chaperones are also essential for the rapid degradation of certain abnormal proteins in both prokaryotes and eukaryotes (16,24). These short-lived proteins appear to associate with DnaJ and other chaperones prior to their degradation (16,24). Since chaperones bind specifically to unfolded polypeptides, they may participate in the recognition of abnormal proteins by the degradative machinery. The present studies were undertaken to determine whether molecular chaperones may also be important in the degradation of regulatory proteins.To examine this possibility, we studied whether a DnaJ homolog in Saccharomyces cerevisiae, Ydj1, functions in the rapid ubiquitin-dependent breakdown of the G 1 cyclin Cln3. Cln3 is an important regulator of the Start checkpoint in the yeast cell cycle (7). Cln3 associates with and activates p34 CDC28 kinase in early G 1 phase, which leads to the synthesis of other G 1 cyclins (11, 24a, 25), thus allowing the transition from G 1 to S phase. This interaction occurs through the N-terminal segment of Cln3, which contains the cyclin box, a region conserved among different cyclins. Moreover, this N-terminal segment of Cln3 alone is sufficient for the stimulation of the kinase. p34CDC28 in turn phosphorylates Cln3, which triggers its ubiquitination, leading to the rapid degradation of Cln3 (8,28). This critical pho...

show abstract

Section: Discussionmentioning

confidence: 99%

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Yaglom

Goldberg

Finley

et al. 1996

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Subsequent studies were carried out with repressed HRI, as stress-induced activation of repressed HRI in hemin-supplemented RRL occurs independent of changes in heme concentration (18,22,24,(27)(28)(29). Thus, repressed HRI is the form of HRI that is likely to be the most physiologically relevant target for regulation by diffusible gases in vivo.…”

Section: Effect Of No and Co On Protein Synthesis And Eif2␣mentioning

confidence: 99%

The Heme-regulated Eukaryotic Initiation Factor 2α Kinase

Uma

Yun

Matts

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Nitric oxide (NO) has been reported to inhibit protein synthesis in eukaryotic cells by increasing the phosphorylation of the ␣-subunit of eukaryotic initiation factor (eIF) 2. However, the mechanism through which this increase occurs has not been characterized. In this report, we examined the effect of the diffusible gases nitric oxide (NO) and carbon monoxide (CO) on the activation of the heme-regulated eIF2␣ kinase (HRI) in rabbit reticulocyte lysate. Spectral analysis indicated that both NO and CO bind to the N-terminal heme-binding domain of HRI. Although NO was a very potent activator of HRI, CO markedly suppressed NO-induced HRI activation. The NO-induced activation of HRI was transduced through the interaction of NO with the N-terminal heme-binding domain of HRI and not through S-nitrosylation of HRI. We postulate that the regulation of HRI activity by diffusible gases may be of wider physiological significance, as we further demonstrate that NO generators increase eIF2␣ phosphorylation levels in NT2 neuroepithelial and C2C12 myoblast cells and activate HRI immunoadsorbed from extracts of these non-erythroid cell lines.Over 2 decades ago, the heme-regulated inhibitor (HRI) 1 of protein chain initiation in rabbit reticulocyte lysate (RRL) was identified as a heme-regulated protein kinase that phosphorylated the ␣-subunit of eukaryotic initiation factor eIF2 (reviewed in Refs. 1-3). eIF2 delivers Met-tRNA i in a complex with GTP to 43 S ribosomal initiation complexes and is released as a complex with GDP at the completion of the initiation cycle. Recycling of eIF2⅐GDP and the formation of eIF2⅐GTP⅐Met-tRNA i complexes requires the action of the guanine nucleotide exchange factor, eIF2B. Under heme-deficient conditions, HRI is activated and phosphorylates eIF2. Phosphorylated eIF2 avidly binds eIF2B, sequestering eIF2B in a poorly dissociable complex, which subsequently leads to the inhibition of the initiation of translation, as eIF2⅐GDP complexes that are present in excess of eIF2B fail to recycle. Thus, HRI functions to coordinate globin synthesis with heme availability in RRL.The amino acid sequence deduced from HRI cDNA indicates that it is composed of at least five distinct domains (4). The unique N-terminal domain of HRI contains ϳ165 amino acids. The second and fourth domains contain conserved sequence motifs I-V and motifs VI-XI, which comprise the N-terminal and C-terminal catalytic lobes of protein kinases, respectively. The third and fifth domains are also unique, consisting of ϳ140 amino acids that are inserted between the two conserved kinase lobes and ϳ50 amino acids at the C terminus, respectively.HRI is a hemoprotein that contains two distinct of hemebinding sites (5-7). Heme binding to the first site is stable, and remains associated with purified HRI, whereas heme-binding to the second site is reversible and appears to be responsible for the rapid heme-induced down-regulation of HRI activity . We have recently demonstrated that the N-terminal domain of HRI contains the stable heme-binding...

show abstract

“…Previously, we have observed an inverse correlation between the levels of Hsc70 in different preparations of RRL and the sensitivity of those RRL preparations to heat shock-induced inhibition of protein synthesis [14]. We have speculated that Hsc70, possibly in conjunction with other lysate chaperones, plays a role in maintaining protein synthesis in RRL, and that heat-induced accumulation of denatured proteins leads to the titration of Hsc70 function [14,15]. If protein synthesis rate is coordinated with the availability of functional Hsc70, a correlation should be observed between translational shut-off and levels of functional Hsc70.…”

Section: Conditionsmentioning

confidence: 99%

“…Proteins from 10 µl of the apyrase-treated protein synthesis mixes were adsorbed with the nonimmune control (C) or the anti-HRI (I) mAb F in the presence of NaCl/ Tris. The adsorbed fractions were separated by SDS/PAGE, transferred to a poly(vinylidene difluoride) membrane, and western blotted with the 4322 anti-Hsp90/Hsp70 antibody to detect the presence of Hsc70, as described previously [15,25]. HC, heavy chain of IgG.…”

Section: Conditionsmentioning

confidence: 99%

“…In addition, HRI in heme-deficient RRL containing low levels of Hsc70 converts more rapidly to an activation state that is resistant to suppression of its activity by the subsequent addition of hemin [14]. Furthermore, the interaction of Hsc70 with HRI is blocked by the addition of denatured protein, such as reduced carboxymethylated-bovine serum albumin (RCM-BSA), to hemin-supplemented RRL, which correlates with RCM-BSA-induced activation of HRI and inhibition of protein synthesis [15]. Recently, supernatant factor, a purified component of RRL that suppresses the inhibition of protein synthesis when added to heme-deficient RRL, has been identified as a Hsp70 family member [16].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Thulasiraman

Uma

et al. 1998

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The role of the heat-shock cognate protein, Hsc70, in regulating the activity of the heme-regulated eIF-2A kinase (HRI) in hemin-supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF-2A phosphorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42°C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg 2ϩ . The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein-synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg 2ϩ , Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF-4 E from heat-induced dephosphorylation, and its inability to protect translational elongation from GSSG-induced inhibition, respectively. Activation of HRI in heat-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss of the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.

show abstract

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2.alpha. kinase

Cited by 55 publications

References 36 publications

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Heme-regulated Eukaryotic Initiation Factor 2α Kinase

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Contact Info

Product

Resources

About

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2.alpha. kinase

Cited by 55 publications

References 36 publications

The Molecular Chaperone Ydj1 Is Required for the p34CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Heme-regulated Eukaryotic Initiation Factor 2α Kinase

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Contact Info

Product

Resources

About

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation