METODOLOGÍA PLANTEADA PARA EVALUAR LOS RESULTADOS DE

Oxidative stress Osmotic stress Heat stress Membrane Makl/2? Mcs4 MKKK Wis4 Win4 Cytoplasm MKK VVisl MK Styl General Nucleus response ^ Oxidative 5'— ARE —^ stress --- response Osmotic stress SLNl SHOl YPDl SSKl SSK2/2: STEll PBS2 - t® HOC! 5" STRE r o St] smotic stress response

com ponents. Analysis of events upstream of H O Gl are particularly revealing. PBS2 seems to be the only MKK w hich can activate H O G l, how ever, there are two separate inputs from MKKKs. One arm in v o lv e s the use of a tw o-com ponent histidine kinase cascade, sim ilar in n atu re to those d o m in an t in bacterial signalling, which activates two re d u n d a n t MKKKs. The other uses a MKKK previously thought to be a shared com ponent of the m ating and filam entous grow th MK pathw ays. For b o th arm s, the transm em brane com ponent which presum ably acts as the direct osm osensor is known. H ow ever, the precise m echanism by w hich changes in osm olarity are sensed rem ain unknow n.

The discovery of a eukaryotic two-com ponent signalling in p u t to th e H O G l cascade was unexpected, although in recent years tw o -co m p o n en t signalling systems have been found across several kingdom s, in clu d in g archaebacteria (Rudolf 1995), plants (Chang 1993), and in the slime m o ld

Dictyostelium d ic o id eu m (Schuster 1996). Prokaryotic tw o -co m p o n en t

systems, of w hich over 100 have been identified, typically consist of a sensor histidine kinase and an effector protein called the response regulator (Egger 1997). Stim ulation of the sensor results in its dim érisation, and subsequent p h osphorylation of a histidine residue situated near the kinase catalytic dom ain. This phosphate group is then transferred to an aspartic acid residue w ithin the receiver dom ain of the response regulator, activating its effector function, w hich usually involves direct transcriptional control of target genes.

A sim ilar m ulti-step His-Asp phosphorelay takes place in b u d d in g yeast in response to external osmolarity. The transm em brane SL N l histidine kinase initiates the phosphorelay, w hich goes via an in term ed iary (YPDl) to an aspartate residue in the receiver dom ain of the SSKl response regulator (Posas 1996). SSKl acts directly to m odulate the activity of th e MKKKs at the top of the H O G l cascade. Unlike m ost bacterial response regulators, this eukaryotic response regulator is inactive w h e n phosphorylated. A ctivation of HOGl results from a high osm olarity environm ent, w here SLNl is inactive, and the non-phosphorylated SSKl is able to interact w ith the N -term inus of one of two red u n d an t MKKKs,

SSK2 and SSK22. The binding of SSKl stim ulates MKKK autophosphorylation and results in the subsequent activation of the H OGl pathw ay (Posas 1998). The im portance of negative regulation of H O G l by SLN l is dem onstrated by the lethal phenotype caused by deleting the S L N l

gene (resulting in a constitutively active H OGl cascade). This lethality can be rescued by deletion of H O G l or PBS2, or by overexpressing genes encoding the phosphatases that oppose these kinases; PTP2, PTCl, or PTC3

(Maeda 1994 and Maeda 1993) (see below).

In addition to this SLNl dependent pathw ay, a second sensing ro u te is available via a putative transm em brane protein called SHOl (Maeda 1995). SH O l transm its a signal to PBS2 via the MKKK ST E ll (Posas 1997). Interestingly, ST E ll also participates in MK pathw ays required for m atin g and p seudohyphal differentiation (see FUS3 and KSSl below) illu stratin g the point that one MKKK can activate two different MKKs (PBS2 and STE7). H ow ever, even though ST E ll is involved in m ultiple signalling pathw ays, there appears to be no crosstalk betw een the HO G l and m ating pathw ays (Posas 1997). This lack of crosstalk m ay be accomplished by scaffolding proteins w hich link the sensory apparatus to the individual MAPK m odules. In the case of the m ating pathw ay, this function is carried out by the Ste5 protein (see FUS3 below). In the case of the H O G l pathw ay, interaction assays suggest that PBS2 itself m ay facilitate the form ation of a m u ltip ro tein complex which restricts the osmotic stress response to th e H O G l pathw ay (Posas 1997).

Follow ing HOG activation, the separate phosphatase activities of PP2C hom ologs (PTCl, PTC2, PTC3) and two PTPs (PTP2 and PTP3) contribute to kinase pathw ay inactivation (Maeda 1993; W u rg ler-M u rp h y 1997). PTP2 and PTP3 bind and dephosphorylate HO G l, w hereas the PP2C activities m ight be directed at either PBS2 or H O G l (Maeda 1994).

A m olecular understanding of events dow nstream of H O G l is farther from com pletion. To date, no in v i v o substrate has been show n to be directly phosphorylated by HOGl. The transcription factors MSN2 and MSN4 (see STREs in the Introduction of Section 2, and Sum m ary) can

clearly function dow nstream of the H O G l pathw ay, b u t they do not seem to be direct targets for this kinase.

MPKl

MAP Kinase 1/su p p resso r of lyt2 (MPK1/SLT2) was isolated as a dosage dependent suppressor of a tem perature dependent cell lysis th a t results from deletion of the MKKK BCKl. Deletion of M P K l results in th is sam e tem perature dependent lysis phenotype (Lee 1993), w hich is due to defects in cell wall construction (Costigan 1992). MPKl becomes activated d u rin g periods w hen yeast cells are undergoing highly polarized cell grow th (such as during bud form ation and projection form ation after m ating p herom one stim ulation) (Zarzov 1996), and in response to hypotonic or heat shock conditions where the cell wall requires rem odelling (D avenport 1995).

The MPKl MK cascade, m ade up of BCKl, the red u n d an t MKKl and MKK2, and M PKl, has been show n to act dow nstream of protein kinase C (PKCl) (Irie 1993). Additionally, three different laboratories have recently cloned a gene encoding a putative m em brane protein which m ight fit th e role of a sensor molecule for the cell wall stresses that activate this pathw ay. A lternately know n as SLGl Qacoby 1998), HSC77 (Gray 1997), or W S C l

(Verna 1997), deletion of this gene results in sim ilar phenotypes as those resulting from m u tatio n of PKCl, BCKl, and M P K l . Interestingly in p u t from another MKKK to MKK1/MKK2 has been im plied by results show ing that the stim ulation of MPKl that occurs subsequent to p h e ro m o n e signalling depends on PKCl, M K K l and MKK2, but does not require BC Kl

(Buehrer 1997). Similar to m any of the know n MKs, M PKl has been sh o w n to directly bind its MKK, either MKKl or MKK2 (Soler 1995).

O verexpression of M P K l also results in a cell lysis phenotype, and this effect has been used to develop genetic suppressor screens to look for d ow nstream effectors. Two dow nstream transcription factors that h a v e been identified are the MADS-box family m em bers RLMl and SM Pl (W atanabe 1997; D odou 1997); how ever, it has not been established th at these factors are direct targets. Recently, evidence was also published that a

potential in v i v o substrate for M PKl is the SBF complex (M adden 1997). SBF is a heterodim eric complex composed of SWI4/SWI6 that regulates th e G l/S transition by activating expression of the G1 cyclin genes. P hosphorylation by M PKl m ay be im portant for coordinating entry into S phase w ith polarized grow th events, since a num ber of cell w all com ponents are regulated by SBF (Igual 1996).

FUS3

Peptide m ating pherom ones cause haploid S.cerevisiae to arrest m itotic grow th at the G1 phase of the cell cycle, and express m ating specific genes in preparation for conjugation w ith a cell of the opposite m ating type. The m olecular dissection of this behavior has led to one of the best u n derstood signalling cascades, which utilises the MK FUS3 (identified as a gene product w hose loss results in a cell fusion defective phenotype) (review ed in H erskow itz 1995). Binding of pherom one to tran sm em b ran e receptors that are coupled to heterotrim eric G proteins activates m olecules (CDC42, STE20, STE50) just upstream of the MK cascade consisting of S T E ll (MKKK), STE7 (MKK), and FUS3 (MK). An additional MK, KSSl, can function in place of FUS3 in a FUS3 delete, but norm ally controls a n o th e r d evelopm ental cell fate (see below). Activated FUS3 targets a tran scrip tio n factor heterodim er, m ade up of STE12 and M CM l, w hich is a key reg u lato r of m ating specific genes. Additionally, FARl, a cyclin dependent kinase inhibitor, is phosphorylated by FUS3 and m ediates the pherom one induced G1 arrest (Elion 1993).

Phosphatase control of the pherom one pathw ay has also been described. One dual specific phosphatase, MSG5, has been cloned an d show n to negatively regulate the pherom one pathw ay. M S G 5 is transcriptionally induced by the pherom one pathw ay, resulting in FUS3 being only transiently active (Doi 1994). FUS3 can also be dephosphorylated on tyrosine by both PTP2 and PTP3; indeed PTP3 m ay be the m ajo r phosphatase responsible for m aintaining a low basal activity of the kinase (Zhan 1997).

Signalling specificity in this pathw ay is controlled at several levels. A 'scaffolding m olecule', STE5, has been described, w hich sim u ltan eo u sly binds the m ating pathw ay G protein, STEll, STE7, and FUS3, and could function to channel the signal (W hiteway 1995). A dditionally, there is an intrinsic specific affinity betw een the MKK and MK com ponents. B oth FUS3 and KSSl have been show n to form tight complexes w ith STE7 v ia their extrem e N -term ini (see below), while H O G l, M PKl, and ERK2 do n o t (Bardwell 1996). Finally, signalling specificity for FUS3 versus KSSl is partially controlled by an inhibitory activity of FUS3 th at p rev e n ts in ap p ro p riate activation of invasive grow th by the pherom one pathw ay in h aploid cells. Cells which have FUS3 deleted are hyperinvasive, w hile cells containing a kinase-dead FUS3 do not exhibit this inappropriate activ atio n (M adhani 1997). Presum ably, the absence of FUS3 allows KSSl (see below) to be activated by pherom one, resulting in the induction of target genes u tilised in invasive growth.

KSSl

K S S l w as originally identified as an overexpressed kinase suppressor

of an SST2 m utation, w hich allows these pherom one grow th-arrested cells to restart grow th (Courchesne 1989). Until recently, KSSl activity, in th is and additional assays, led to the conclusion that KSSl was acting red u n d an tly w ith FUS3 in the pherom one pathw ay. It is now clear, how ever, that KSSl function is specifically critical for regulating filam entous grow th. Diploid S.cerevisiae starved for nitrogen undergo a dim orphic transition to form pseudohyphae composed of in v a siv e filam ents of elongated cells; a related phenom enon, invasive grow th, occurs in haploid cells grown on rich m edia (Roberts 1994). B oth phenom ena require upstream components that are also used by the m atin g pathw ay (including STEll and STF7) as well as the d o w n stream transcription factor STF12 that functions in m ating. H ow ever STF12 activated by KSSl associates specifically w ith the transcription factor TFCl on filam entation response elem ents to activate filam entous grow th genes

(M a d h a n i 1997). Paradoxically, b o th FUS3 and KSSl are d isp en sab le for f ila m e n ta tio n .

A m o d e l th a t m ig h t explain this c o n u n d ru m is tak in g sh ap e, n o w th a t it has b een sh o w n that, like FUS3, KSSl has d istin ct p o sitiv e a n d n e g a tiv e sig n allin g roles. The n eg ativ e role of KSSl is p a rtic u la rly in te re stin g . Inactive KSSl directly b in d s the tra n scrip tio n factor STE12 via a b in d in g site th a t is located on its activ atio n loop, a n d in h ib its STE12 m e d ia te d a c tiv atio n of filam e n to u s g ro w th genes (B ardw ell 1998). T h is in h ib itio n is likely to in v o lv e e ith e r re c ru itm e n t or stab ilisatio n of a co m p lex of STE12 w ith the in h ib ito rs D IG l an d DIG2 (Cook 1996; also iso lated as RSTl an d RST2 (Tedford 1997)). R em oval of these in h ib ito rs is su fficien t to activ ate STE12 d e p e n d e n t genes (i.e. DIG m u ta n ts do n o t re q u ire e ith e r FUS3 or KSSl for filam ento u s grow th). F ollow ing a signal for fila m e n ta tio n , STE7 p h o sp h o ry la tes KSSTs activ atio n loop, th e re b y T ^

re d u c in g th e b in d in g of STE12 (B ardw ell 1998). A d d itio n ally , su b se q u e n t p h o s p h o ry la tio n of STE12 by KSSl (not directly p ro v e d in v i v o ) likely acts to p o sitiv e ly activate STE12, possibly by d e sta b ilisin g K S S l/S T E 12 /D IG 1 /D IG 2 and freeing the STE12 activ ato r from in h ib itio n . As re sea rc h on o th e r MK sig n allin g p ath w ay s co n tin u es, it w ill be in te re stin g to see if this strategy u sin g the in d u cib le release of in h ib ito r m o lecu les to allow activ atio n is u n iq u e, or m ore g en erally used.

S M K l

M eiotic d e v e lo p m e n t in S.cerevisiae is ch aracterised by an o rd e re d p a tte rn of ex p ressio n of early, m id d le, m id-late, a n d late s p o ru la tio n genes. S M K l is a n MK th a t is re q u ire d for spore m o rp h o g e n e sis an d is ex p ressed as a m id d le s p o ru la tio n gene. S M K l m R N A , w h ich is n o t detectable in v e g e ta tiv e cells, is d erep ressed at least 200-fold ju st p rio r to p ro s p o re e n c lo su re (K risak 1994). N e ith e r u p stre a m activato rs n o r d o w n s tre a m ta rg e ts for SM K l h av e been identified to date.

s . p o m b e M K s

In co n trast to the n u m e ro u s m a m m a lia n , and five b u d d in g yeast MKs, o nly th ree MKs h av e been id en tified in S . p o m b e th u s far: S ty l, S p k l, an d P m k l (see Figure lA an d Figure IB). The m ost u n d e rs to o d e n zy m e is S ty l, the focal MK in this thesis. R em arkably, this MK is crucially in v o lv e d in a v e ry w id e ran g e of sig n allin g p h e n o m e n a . S p k l is the MK re s p o n d in g to p h e ro m o n e signals in S . p o m b e , w hile P m k l's fu n c tio n is p o o rly u n d e rs to o d , b u t it re sp o n d s to signals used for cell w all c o n s tru c tio n a n alo g o u s to M PK l of S.cerevisiae. The three cascades re p re se n te d by th e se MKs a p p e a r to be fu n ctio n ally distinct. I will briefly su m m a rise c u rre n t k n o w led g e of these kinases below .

Styl

S ty l is a m u ltifu n c tio n a l SK of S.pombe th at becom es activ ate d