EVALUACIÓN DE LOS ÍNDICES DE GESTIÓN DE LA CALIDAD DEL

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In contrast, S.cerevisiae proteins w ith hom ology to the m a m m a lia n API (c-Jun) bZip dom ain have long been dem onstrated to be involved in stress signalling. The prototypical bZip protein GCN4 can bind to a canonical A PI cis elem ent (ARE) and regulates a large num ber of genes induced following am ino acid starvation (Hope 1985). In addition, GCN4 m ediates a protective response against irradiation by ultraviolet light (UV) sim ilar to a function of m am m alian API (Engelberg 1994). A nother group of yeast A PI proteins (YAPs) m ake up a family of eight m em b ers (Fernandes 1997), two of which, Y API and YAP2, have been show n by a num ber of laboratories to be critical for signalling oxidative stress and regulating drug resistance (Moye-Rowley 1989, Kuge 1994). Interestingly, Y API and YAP2 uniquely have an additional conserved region, the cysteine rich dom ain (CRD), that has been recently show n to function in regulated nuclear translocation of these proteins following oxidative stress (Kuge 1997).

The use of these API response elem ent (ARE) binding tran scrip tio n factors u n d er various oxidative stresses has strong parallels to the fu n ctio n of c-Jun in oxidative stress in m am m alian cells (Karin 1997), and gives credence to the hypothesis of conservation of both functional and structural aspects of stress signalling. Surprisingly, however, a role for MK cascades i n m odulating YAP activities has not been discussed (or at least reported). Clearly, either positive or negative data concerning this p otentially conserved m echanism , deserves future exam ination.

S.pombe bZip Factors

As I began this thesis work, three bZip transcription factors had been identified in S.pombe. During the S.pombe sequencing project a gene encoding a bZip dom ain w ith 53% identity to that of m am m alian ATF2 w as identified. This A tfl protein factor was found to be required for S . p o m b e

extracts to bind to a CRE (the same CRE utilised by ATF2) (Takeda 1995; Kanoh 1996; also see Jones 1989). Perl (pombe CRE response factor) had also been show n to bind to this CRE in a heterodim er complex w ith A tfl (W atanabe 1996; Kanoh 1996). Finally, Papl (pombe API), had been

identified as having sequence hom ology, and sim ilar binding specificity, to the m am m alian and b udding yeast API factors (Toda 1991) (see Figure 4).

Interestingly, deletion of each of the genes encoding these th ree proteins was associated w ith stress signalling phenotypes. A tfl deleted

(Aatfl) cells failed to enter stationary phase upon n u trien t stress and w ere

described to be cold sensitive (Takeda 1995). In addition, these cells also could not respond appropriately to nitrogen stress; Aatfl cells fail to arrest properly in G1 (indeed atfl was also cloned as G1 arrest defective gad7

(Kanoh 1996)) and have both m ating and meiotic defects (Takeda 1995). Perl deleted cells (Apcrl) had similar, although weaker, phenotypes (W atanabe 1996). Cells lacking Papl (Apapl) failed to survive various oxidative stresses, and exposure to a num ber of different toxic chemicals (Toda 1991).

An exam ination of the S.pombe genome (approxim ately 65% com pleted as of 10/98) using a TBLASTN search (Altschul 1990)(BLAST search perform ed using the N ational Center for Biotechnology In form ation's BLAST WWW Server and the Sanger Centre S . p o m b e

Sequencing Project BLAST WWW server) w ith the bZip dom ain of A tfl (or ATF2), retrieves only four genes w ith significant hom ology (P value<4xe- 12); atfl, pcrl, atfZl, and cbpl (see Figure 4). a t f l l was identified as a h ig h copy genetic suppressor of styl deleted celTs m ating defects (Shiozaki 1996) d u rin g the course of my studies. However, deletion of this gene failed to generate any observable phenotype. Similarly, the gene cbpl, identified by sequence analysis of the S.pombe genome, fails to dem onstrate a recognised ph en o ty p e u p o n deletion (Millar, Toone, Samuels observations). A sim ilar BLAST search using the bZip dom ain of Papl (or Y API) identifies only

pa pl.

It is quite possible that additional bZip transcription factors will be identified as the S.pombe genome sequencing is finished, but it is striking th at this organism utilises just a small num ber of these m olecules. It is particularly interesting that S.pombe has only one A Pl-like m olecule, considering that S.cerevesiae utilises so many. The fact that these yeast proteins have sim ilar DNA binding dom ains as im portant m a m m a lia n

factors m ay be evolutionarily significant, and is certainly useful for testing functional m odels.

Scope of Stress Studies

This thesis details my investigations into stress signalling in fission yeast. For the purposes of this work, stress is broadly defined as adverse conditions to w hich cells m ust respond in order to continue n o rm a l grow th. This includes both physical stress (such as tem perature and salt stresses) and metabolic stress (such as n u trien t stress). Cells have bo th im m ediate responses and long term adaptations to changes in environm ental conditions, b u t my focus will be on the im m ediate response to various physical stresses. W hile these im m ediate responses can occur at m any levels (post-translational dow n to transcriptional), I will concentrate on transcriptional responses that are generated following p h o sp h o ry latio n of pre-existing protein kinases and transcriptional factors. A dditionally, these transcriptional responses can be in the form of up-regulation or dow n-regulation, and I will describe only genes that are up-regulated in response to stress. M any studies have concentrated on transcriptional activation by positive factors, but more has come to light recently concerning the role that 'relief of repression' has as an activating m echanism . In S.cerevisiae the loss of D N A -binding molecules such as M IG l, and RNA polym erase-interacting co-repressors like TU Pl, h a v e illustrated an im portant role for repression of response genes. I w ill describe research on this aspect of gene regulation, but will concentrate o n the action of transactivating molecules.

Finally, m y choice of genes to analyse has prim arily been in fo rm ed by w h at is know n about responses in S.cerevisiae. This tests th e conservation hypothesis directly, and makes use of the fact that stress response genes are well characterised in this organism. I will leave th e discussion about stress response genes to Section 2 of the thesis, and w ill first detail m y w ork concerning the protein factors involved in signalling stress.

1.2: RESULTS: A tfl Activation by Styl

Rationale for Styl / A tfl Experiments

This w ork grew out of studies initiated by Tadayuki Takeda, w hile in the laboratory of Nic Jones (Takeda 1995), and the observation that cells deleted for A tfl (Aatfl cells) were sensitive to osmotic stress. This phenotype, along w ith the inability to m ate and form spores, and the p oor viability of cells entering stationary phase, were very sim ilar to phenotypes previously dem onstrated for cells deficient in either Styl (As tyl cells) or

W i s l ( A w i s l cells) (Millar 1995; Shiozaki 1995; Stettler 1996). Styl is a n

S.pombe MK, and W isl is its MKK (described in S.pombe MKs, above). O ne

could expect that phenotypes resulting from inactivation of a MK cascade w ould be shared by cells lacking the critical target substrates for the MK, and it seem ed possible that A tfl was such a substrate. In addition, the sequence hom ology and com m on DNA binding characteristics of A tfl com pared to ATF2, a know n transcription factor substrate for SK signalling cascades in m am m alian cells, presented a rationale for an exam ination of th e hypothesis that A tfl was a substrate for a conserved SK cascade in S.pombe.

Styl Phosphorylates A tfl in v itr o

In order to test this hypothesis, I first needed to generate m aterial for use in an in vitro kinase assay. A plasm id containing an inducible prom oter (through the use of the sugar analogue IPTG), followed by D N A encoding an N -term inal fusion betw een glutathione S-transferase (GST) and A tfl w as used. This plasm id was transfected into the bacteria E. coli,

and GST A tfl expression was induced by the addition of IPTG. GST A tfl was subsequently purified using standard techniques (see M aterials and Methods). The prepared GST A tfl was som ew hat unstable, resulting in th e appearance of the full length molecule (running at a m olecular w eight of approxim ately 100 kilodaltons) and several sm aller species of breakdow n products (see Figure 5C). The final purified m aterial b ound to g lu ta th io n e Sepharose beads was tested as an in vitro substrate for im m u n o p recip itated Styl.

A cell line containing a hem e agglutinin (HA), and six h istid in e (6His), tagged Styl was obtained from our collaborator Jonathan M illar for use in the kinase assay. This cell line contains a d isruption of th e endogenous gene encoding Styl, and a plasm id expressing the C -term inally tagged Styl that is repressed by the am ino acid th ia m in e

(Rep41stylHA6His). This plasm id allows for inducible expression of Styl

and rescue of the Astyl phenotypes (Millar 1995). These cells were grow n to m id-log phase in the appropriate m inim al m edia, and given a salt shock w ith the addition of KCl to a final concentration of 0.7M. Samples w ere rem oved at different time points following salt addition, and lysates w ere prepared (see M aterials and Methods).

The in vitro kinase assay was perform ed by im m unoprécipitation (IP) of

StylH A 6H is, using antibodies specific for the HA tag, followed by extensive w ashing, and incubation w ith GST A tfl in a kinase reaction buffer containing radiolabeled ATP (see M aterials and M ethods). The reaction m aterial w as ru n out on a polyacrylam ide gel (PAGE) and then either dried dow n and exposed to X-ray film, or transferred to a m em brane for W este rn blotting w ith an antibody specific for HA. The results from this assay are show n in Figure 5A and Figure 5B.

Figure 5A illustrates the inducible (repressible) expression of StylH A 6H is in the Rep 41 sty lH A6 H is containing Astyl cells that w ere used th ro u g h o u t m y thesis work. StylHA6H is is present in the lysates w h e n thiam ine (T) is absent (seen as a 45 kilodalton band just u n d er the 50 kilodalton heavy chain IgG), and its abundance does not change during th e experim ent. In contrast, there is a very tight shut off of StylH A 6H is expression in the presence of thiam ine, w ith no detectable protein even at long exposure times. The use of the 'm iddle range' Rep41 expression vector

(Repl transcribes the highest level, and Rep81 the lowest) was appropriate,

as was recently determ ined w hen a cell line containing s ty l H A ô H i s

integrated dow nstream of its ow n prom oter became available (Shieh 1997), and was found to express equivalent protein levels as R e p 4 1 s t y lH A 6 H i s

The results from the IP kinase assay using these lysates are show n in Figure 5B, and clearly dem onstrate that GST A tfl can serve as a substrate for Styl. Phosphorylation of A tfl depends on both the presence of Styl in th e lysate (i.e. no signal in the presence of thiam ine after salt treatm ent), and on S ty l's activation by salt shock. It is also im portant to note that activ atio n of Styl, as m easured by phosphorylation of A tfl, is both rapid and tran sien t. This result is m atched by Nickel W estern experim ents (purification of Styl via the 6His tag binding to a nickel affinity m atrix, followed by W este rn blotting) using these cell lysates, w here StylH A 6H is's in v i v o activity is assessed using antibodies against phosphotyrosine (see Figure 4B in W ilkinson 1996, attached). Specificity controls for the kinase assay w ere perform ed, including the use of GST fused to other proteins co n tain in g potential phosphoacceptor sites (see Figure 7C for example), and using free A tfl following cleavage of the GST m oiety (data not shown).

Several m etozan MKs have been dem onstrated to function by first binding the substrate at an epitope distinct from the phosphoacceptor site, and only subsequently phosphorylating the substrate. I tested w hether Styl conserves the use of this m echanism by mixing GST A tfl, still im m obilised on Sepharose beads, w ith the lysates previously described. After several hours the GST A tfl beads were spun down, extensively w ashed (in order to rem ove all cellular proteins that could not bind to the GST A tfl), and th e n placed in a kinase reaction m ixture w ith radiolabeled ATP, as before. T he results from this 'pulldow n kinase' experim ent are show n in Figure 5C.

Again A tfl was phosphorylated quickly and transiently, in a m a n n e r that depended on the presence of Styl in the lysate. The clear in te rp retatio n of this experim ent is that A tfl is not only a specific substrate for activated Styl, b u t th at there is tight binding of A tfl to Styl (resistant to m u ltip le rounds of washing) before it is phosphorylated. Thus, there is a m echanistic conservation of MK activity tow ards its substrate betw een this yeast MK and vertebrates MKs (see 1.1. Transcription Factor Substrates).

S ty l is an SK

In m etazoan cells, MKs fall into two m ajor classes, ERKs and SKs. The n atu re of the amino acids in the kinase activation loop have been good predictors for this classification; TEY for ERKs, and TGV or TRY for SKs (where T stands for threonine, Y for tyrosine, E for glutam ate, G for glycine, and P for proline). Styl has a TGY in its activation loop (see Eigure IB) and th u s w ould be predicted to be a SK. Its sequence is 86% identical to that of H O G l, the osmoticly activated SK of S.cerevisiae. In addition, Styl activity is induced by a salt shock (as dem onstrated above), and deletion of styl

gives an osm osensitive phenotype (Millar 1995).

H O G l responds only to osmotic shock (see 1.1. HOGl), w h ile m am m alian SKs are activated by a large variety of stresses in addition to salt shock. Thus, it w as of interest w hether Styl is exclusively responsive to osm otic shock, or is m ore like the m am m alian SKs. Kinase assays, u sin g GST A tfl as a substrate, dem onstrated that Styl is activated not only by osmotic stress, but also by heat stress and oxidative stress (data not show n). In addition, Astyl cells are phenotypically sensitive to heat stress, oxidative stress, and to heavy m etals and toxins (see Figure 1 in Toone 1998, attached). A num ber of published reports have also concluded that Styl is responsive to m any stress stim uli (Degols 1996; Kato 1996; Degols 1997; Shieh 1997).

SKs have an additional distinguishing feature in that there are chemical inhibitors that have been show n to exclusively interact w ith SK's and not w ith ERKs. I used one such inhibitor, SB203580, in the in v i t r o

kinase assay w ith A tfl, to see if Styl had a sim ilar inhibition profile. A chem ical inhibitor specific to the ERK pathw ay, PD98059, was used as a control. The SB203580 inhibitor completely blocked Styl activity at a concentration of 2 m icrom olar (similar to that used to block m a m m a lia n SKs), w hereas, S tyl's activity was unaffected by the PD98059 inhibitor at concentrations u p to 20 m icromolar (data not shown).

Therefore, by the criteria of sequence sim ilarity, com m on activating stim uli, and specific chemical inhibition, Styl is an S.pombe SK.

Mapping A tfl's Styl Binding Site

I next began to determ ine w hich residues of A tfl are im portant for binding Styl, and which act as phosphoacceptors. A first com parison of th e A tfl sequence upstream of the bZip dom ain w ith m am m alian ATF2 or c- Jun sequences revealed little useful homology. Therefore, I began a n unbiased search for these activities on A tfl.

I first constructed GST fusions w ith various portions of A tfl for use in both TP kinase' and 'p ulldow n kinase' assays. The various constructs used are show n in Figure 6A, and the kinase assays are show n in Figure 6B (using lysates before and after 15 m inutes of 0.7M KCl addition). T he Coom assie stained gel is show n for comparison. The results in the figure used im m unoprecipitated StylHA6His for the kinase assay, but identical results are found using a GST A tfl pulldow n kinase assay, A tfl contains 11 potential MK phosphorylation sites, defined as being either serine o r th reo n in e residues, followed by a proline. A GST fusion w ith the N - term inal portion of A tfl, from residue 1-294, contains 10 of these p o ten tial sites and is sufficient to pulldow n activated Styl, as m easured by its phosphorylation. A com parison of the extent of radioactive p h o sp h ate incorporated in this portion of A tfl w ith similar am ounts of the full len g th fusion m olecule (wt) show approxim ately equal levels. Next, th ree overlapping fusions covering this N -term inus were made: one co n tain in g residues 1-148, one w ith residues 81-195, and the third containing residues 190-294. The fusion molecule containing amino acids 81-195 was completely