Efecto del tiempo de proceso para remoción de arsénico de

am ino or carboxy term inus of RhoGEFs can lead to increased activity in vivo; m any truncated RhoGEFs have been isolated in screens for transform ing genes (Cerione and Zheng 1996). Studies in C hapter 4 examine transform ation by the RhoGEFs dbl and mNETl.

1.5.3 Rho subfamily small G proteins in S. cerevisiae

In S. cerevisiae Rho subfam ily small G proteins co-ordinately regulate a w ide range of processes. S. cerevisiae has five Rho family small G proteins, the best characterised are CDC42 and R H O l. CDC42 and R H O l are an essential genes th at are involved in the co-ordinated reg u latio n of b u d form ation, actin organisation, and regulation of cascades of kinases th at signal to the nucleus. Cdc42 co-ordinates the changes in actin stru ctu res, cell sh ap e an d gene e x p re s s io n u p o n e x p o su re to p h e ro m o n e . C dc42 re g u la te s th e S t e l l / 7 / F u s 3 / K s s l kinase cascade w hich is req u ired for resp o n d in g to pherom ones and the sw itch to invasive grow th u n d e r n u trie n t d eprivation conditions (similar to the MEK/ERK cascade Section 1.2.2 and Figure 1.1 (Simon

et al. 1995; Zhao et al. 1995)). Cdc42 regulates these kinases through activation of

the kinase Ste20. Ste20 binds directly to Cdc42 and this interaction is required for invasive grow th and efficient m ating responses (Peter et al. 1996). R h o l co­ ordinates regulation of cell wall synthesis w ith budding and also is involved in signalling cell wall and environmental stress to the nucleus (see Figure 1.9). R hol regulates cell wall biosynthesis via interaction w ith pl-3glucan synthase and signals cell wall stress to the nucleus via the kinase Pkcl (Drgonova et al. 1996; Q adota et al. 1996; Nonaka et al. 1995). The direct interaction of Rhol w ith Pkcl is required for regulation of the B c k l/M k k l/2 /M p k l kinase cascade in response to en v iro n m en tal stresses. This kinase cascade regulates the activity of the transcription factors, R lm l, N hp6A and N h p6B (W atanabe 1995; Costigan et al.

1994). Pkcl is also required for bu d form ation (Levin et al. 1990). R hol and Cdc42 both interact w ith the formin homology containing protein Bnil, which is required for bu d form ation and m ay regulate the actin cytoskeleton by binding profilin. Both Cdc42 and R hol regulate several aspects of cell behaviour and ensure these processes are co-ordinated (see Figure 1.9).

In response to pherom ones Cdc42 is activated via the activated receptor triggering activation of heterotrimeric G proteins and the subsequent binding of the P subunit, Ste4, to the Cdc42 GEF, Cdc24 (Simon ef al. 1995; Zhao et al. 1995). The Ras2 sm all G protein m ay activate Cdc42 u n d er conditions of n u trien t deprivation, the molecular details of this are unclear (Mosch 1996), b u t it m ay be analogous to the activation of Racl by ras in m am m alian cells (see below). The upstream regulation of R hol is also unclear. Two Rhol GEFs have been isolated,

Pheromones Ste20 Psuedo- hyphae Ste4 Cdc24

T

f

▼

t

t

t

T

Cell wall

Figure 1.9: Role of R hol and Cdc42 in yeast. The processes regulated by Rhol and Cdc42 in S. cerevisiae are outlined together with their upstream regulators and effector molecules. Both Rhol and Cdc42 regulate cell growth, budding, the actin cytoskeleton and transcription factors (underlined).

R om l and Rom2; these appear to be regulated by phospholipids and potentially the lipid kinase Tor2 (Ozaki et al. 1996; Schmidt et at. 1996).

1.5.4 Rho subfamily of small G proteins in mam m alian cells

The twelve know n members of the Rho subfamily can be further divided on the basis of prim ary sequence (see Figure 1.7A). Besides RhoA, the best studied m am m alian m embers of the Rho subfamily are Racl and Cdc42. The study of the function of Rho subfamily small G proteins in m am m alian cells has relied heavily on the use of m u tan ts th at are defective in GTP h y d ro ly sis an d bin d constitutively to GTP, G14>V and Q63>L in RhoA, (green asterisk in Figure 1.8A) or m utants only able to bind GDP, T19>N in RhoA, (red asterisk in Figure 1.8A). Studies of RhoA have also utilised the bacterial toxin C3 transferase, w hich ribosylates amino acid 41 of RhoA and inactivates RhoA b u t not Racl or Cdc42 (purple asterisk in Figure 1.8B). Unlike ras, techniques do not exist to directly

m easu re the GTP lo ad in g of RhoA. I have m ade extensive use of the

constitutively G TP-bound RhoA m u ta n t RhoAV14 and expression of C3 transferase (also referred to as C3 toxin) to analyse the function of RhoA and its effectors.

Function of Racl

Like their yeast counterparts, the m am m alian Rho subfam ily sm all G proteins regulate a w ide range of processes. R acl is required for the generation of p erip h eral polym erised actin structures term ed lam ellipodia in response to PDGF, insulin, and activated ras in fibroblasts. Lam ellipodia m ay detach from the substratum and fold back into the cell generating m em brane ruffles. GTP bound Racl is sufficient to produce lamellipodia and the clustering of vinculin at the cell periphery in the absence of any stimuli (Ridley et al. 1992; Nobes and Hall 1995). The form ation of lamellipodia is a precursor to cell m igration events, and Racl is required for cell m igration in several experim ental system s (Van Aelst 1997).

Racl is capable of regulating the activity of a num ber of transcription factors. C onstitutively active Racl can activate the MAP kinase kinase SEK w hich can phosphorylate and activate the JNK/SAPK MAP kinase. JN K/SA PK can then phosphorylate c-jun and prom ote its transcriptional activation (Figure 1.1 - M inden et al. 1995; Coso et al. 1995). Several kinases, including PA K l, MLK2 & 3, and MEKK 1 & 4 have been implicated in the regulation of SEK by Racl; however the details of how Racl activates SEK are unclear (Van Aelst 1997). GTP-bound Racl can also activate the transcription factors SRF and NFkB (Hill et al. 1995;

Effector Binds Function PAKl/2/3* MLK2/3* MEKK4* IQGAP pp70S6 kinase MRCK* WASP* PORI PI-5-K p67phox POSH Cdc42, Racl Cdc42, Racl Cdc42, Racl Cdc42, Racl Cdc42, Racl Cdc42 Cdc42 Racl Racl, RhoA Racl Racl

Kinase, activation of JNK/SAPK pathway

Actin depolymerisation and focal adhesion disassembly Kinase, activation of JNK/SAPK pathway

Kinase, activation of JNK/SAPK pathway GAP

Ribosomal subunit kinase Filopodia formation Actin organisation Lamellipodia formation Actin polymerisation NADPH oxidase complex

JNK/SAPK pathway activation, NFkB activation

Perona et al. 1997). The relevance of the regulation of transcription factors by Racl is not clear; Racl does not appear to be required for the regulation SRF. In phagocytic cells Racl associates w ith the superoxide production apparatus and in Hela cells activated Racl increases the levels of reactive oxygen species (Van Aelst 1997). It is possible that the effect of Racl of NFkB activity is m ediated by reactive oxygen species Qoneson and Bar-Sagi 1998). Regulation of both reactive oxygen species and NFkB requires the insert loop of Racl.

Racl is required dow nstream of PI-3-K for ras transform ation and activated Racl can co-operate w ith Raf-1 to cause cell transform ation (Qiu et al. 1995a). GTP-

bound Racl can cause quiescent Swiss 3T3 cells to enter the cell cycle (Olson et al.

1995). R egulation of transcription by Racl does not appear to be linked to transform ation (W estwick et al. 1997). Several m olecules have been isolated w hich bin d preferentially to GTP-bound R acl and m ay potentially m ediate Racl's effects (see Table 1.4). PORI, PIP-5-Kinase and IQGAP m ay be involved in cytoskeletal regulation; the kinases PAK, MEKK4, and MLK3 are potentially involved in activation of JNK/SAPK and c-jun (Van Aelst 1997).

Activation of Racl can be m ediated by PI-3-K either acting dow nstream of ras or receptor tyrosine kinases. The PI-3-K substrate, PI(4,5)P2, inhibits the activity of the Racl exchange factor, Vav, whereas the PI-3-K product, PI(3,4,5)P3, prom otes the activity of Vav in vitro (see Figure 1.10 - H an et al. 1998). The regulation of Racl by PI-3-K may be analogous to the regulation of R hol by the lipid kinase

Tor2 in S. cerevisiae (see above). In addition, tyrosine phosphorylation of Vav by

Lck is required for its activity tow ards Racl; this provides another level of regulation of Racl activity (Han et al. 1998). Several other Racl GEFs have been identified including Trio, Bcr, Abr and Sos. Sos can act as a GEF for both ras and Racl through different domains. Racl GEF activity of Sos is regulated by ras in PI-3-K dependent m anner, potentially through the binding of phospholipids to an amino term inal PH dom ain in Sos (see Figure 1.10 - N im nual et al. 1998). Thus Sos and Vav are potential mechanism by which ras and PI-3-K can activate Rac-1.

Function of Cdc42

Cdc42 regulates the form ation of actin-rich spike structures term ed filopodia, these are precursors to the form ation of lamellipodia and in some cases m igration (Van A elst 1997). In m acrophages filopodia probably p lay a role in the phagocytic process. Myotonic D ystrophy Kinase-Related Cdc42-Binding Kinase (MRCK) binds to Cdc42 but not Racl or RhoA in a G TP-dependent m anner and

can enhance the formation of filopodia by Cdc42V12 (see Table 1.4 - Leung et al.

1998). Also implicated in the regulation of actin structures is W iskott Aldrich 49

Syndrom e Protein (WASP). Like R a d , Cdc42 can regulate c-jun th ro u g h the JNK/SAPK pathw ay though it is not clear which effector is involved (Van Aelst

1997). GTP-bound Cdc42 can increase the transcriptional activity of SRF and NFkB, b u t the significance of these findings is as yet unclear (Perona et al. 1997; H ill et al. 1995). GTP-bound Cdc42 can trigger entry into the cell cycle of quiescent Swiss 3T3 cells and Cdc42 m utants that cycle betw een GDP and GXP binding at an increased rate can transform NIH3T3 cells (Olson et al. 1995; Lin et al. 1997). Cdc42 binds to m any of the same target molecules as R a d , how ever Cdc42 is unable to bind PORI and PIP-5-K, the functional significance of m any of these interactions is unclear (see Table 1.4 - Van Aelst 1997).

Function of RhoA related small G proteins

RhoA regulates a wide range of processes including the form ation of actin fibres, focal adhesions and transcriptional regulation (the function of RhoA w ill be discussed in detail in Section 1.6.1.). R ndl, Rnd2 and R nd3/R hoE all antagonise

RhoA function and prom ote the loss of actin fibres and cell rounding (Nobes et al.

1998). U nusually R n d l, Rnd2 and Rnd3/RhoE are unable to bind GDP and are constitutively b ound to GTP. RhoB and RhoD localise to early endosom es suggesting they m ay a play role in endocytosis (Mellor et al. 1998; M urphy et al. 1996). O verexpression of constitutively active RhoD perturbs both endosom e m otility and fusion.

Crosstalk betw een Rho subfamily small G proteins

In some system s Cdc42 m ay be able to activate R a d , w hich can subsequently activate RhoA. In Swiss 3T3 cells GTP-bound Cdc42 prom otes the form ation of R a d dependent lam ellipodia and GTP-bound R a d prom otes RhoA dependent stress fibres (Nobes and Hall 1995). This suggests some sort of functional cascade of Rho subfamily proteins; Cdc42 activates R a d , which in tu rn can activate RhoA (see Figure 1.10). Activation of R a d dow nstream of Cdc42 m ay involve the PAK interacting nucleotide exchange factor (PIX). GTP-bound Cdc42 can bin d to PA K l, which can in turn interact PIX. The interaction w ith PIX m ay prom ote the GEF activity of PIX tow ards both R a d and Cdc42 (M anser et al. 1998). The activation of RhoA by R a d may involve leukotrienes. GTP-bound R a d increases the production of arachidonic acid, possibly through phospholipase A2, which is s u b s e q u e n tly m etab o lised to leu k o trien es (P ep p elen b o sch et al. 1995). L eukotrienes can trigger Rho d ep en d en t actin stress fibre form ation, the m echanism by which leukotrienes activate RhoA is unclear.

Bradykinin CSF i I I PDGF # ♦ 0

%

T

t

T

T

►

♦

t

♦

Lam ellipodia, JNK Stress fibres, SRF

Figure 1.10: Upstream regulation of and crosstalk between Cdc42, Racl and RhoA in mammalian cells. GEFs are shown in green. Upstream regulation of Cdc42 is currently unclear. Upstream regulation of Racl can be mediated by the Sos and Vav, which can both be regulated by PI-3-K, and potentially by PIX, which may act downstream of Cdc42. Upstream of regulation of RhoA can be mediated by pllSRhoGEF acting downstream of Gal3, and potentially by the Racl regulated production of leukotrienes (L'trienes). Unbroken lines indicate direct regulation, dashed lines indicate indirect regulation.

1.6 The RhoA small G protein

1.6.1 Upstream regulation of RhoA

RhoA can be activated by m itogens such as LPA and bom besin. These act th ro u g h seven transm em brane receptors w hich activate h eterotrim eric G proteins. The generic activator of heterotrim eric G p ro tein s, alu m in iu m tetraflouride ion, and the activated G a l3 subunit can activate RhoA dependent processes (Van Aelst 1997). RhoA is directly activated by RhoAGEFs; how ever while m any RhoAGEFs have been identified relatively little is know n about how they are regulated, p i 15 RhoGEF binds directly to GTP-bound G a l2 and G a l3 through a GAP dom ain that is also found in RGS proteins and prom otes GTP hydrolysis by G a l2 and G al3 . In addition, the binding of pll5R hoG EF to G a l3 prom otes the nucleotide exchange factor activity of p i 15 RhoGEF tow ards RhoA (see Figure 1.10 - H art et al. 1998; Kozasa et al. 1998). This is differs from the activation of Cdc42 by heterotrim eric G proteins in S. cerevisiae; in this case the Cdc42GEF, Cdc24, binds to the p subunit of the G protein. GTP-bound RhoA can bind directly to the p subunit of heterotrimeric G proteins, though this is unlikely to be involved in the activation of RhoA as GTP-bound RhoA is already active (Alberts et al. 1998a). Tyrosine phosphorylation of GEFs m ay im portant in the activation of RhoA; tyrphostin, blocks LPA- but not RhoA- induced stress fibres (Ridley and Hall 1994). In addition to external stimuli, RhoA can be activated by activated Cdc42 and Racl in Swiss 3T3 cells (see above). H ow ever, crosstalk betw een Cdc42, Racl and RhoA has not been observed in signalling to SRF in NIH3T3 cells (Hill et al. 1995).

1.6.2 Regulation of the cell morphology and adhesion

Activation of RhoA leads to the formation of long bundles of actin fibres, term ed stress fibres, and the clustering of vinculin, talin, integrins and other cytoskeletal com ponents in focal adhesions, which contact the extracellular matrix (see Figure 1.4 and section 1.3.2 - Yamada and Geiger 1997). Quiescent Swiss 3T3 cells lack stress fibres and focal adhesions; GTP-bound RhoA is sufficient to induce these structures and is required for their induction by LPA and bom besin (Ridley and H all 1992; Van Aelst 1997). RhoA prim arily prom otes the b u n d lin g of pre­

existing F-actin into fibres through actin-myosin interactions and not the de novo

polym erisation of G-actin into F-actin (Mackay et al. 1997). H ow ever, results presented in C hapter 4 suggest that RhoA m ay prom ote actin polym erisation. A ctin-m yosin interactions prom oted by RhoA m ay be im p o rtan t for cell

contractile events induced by RhoA (Uehata et al. 1997). In addition to regulating

focal adhesions RhoA also regulates cell-cell adhesion through cadherins. In 52

MDCK cells RhoA and Racl prom ote the form ation of cadherin m ediated cell contacts (Braga et al. 1997); how ever in sm all cell lu n g carcinom a cells in activ atio n of Rho p ro tein s by C3 p ro m o tes E -cadherin m ed iated cell aggregation (Tokman et al. 1997).

RhoA plays a role in regulating the contraction of sm ooth m uscle cells. Two m ain pathw ays can cause sm ooth m uscle cells to contract: large increases in extra-cellular calcium can trigger contraction and m ore m oderate increases in calcium can sensitise the cells to contraction induced by agonists, including phenylephrine, histam ine and serotonin. GTP7S can mimic the effect of these

agonists suggesting that G proteins are involved. Inactivation of Rho proteins usin g bacterial toxins blocks GTPyS in duced contraction a n d this can be overcome by GTP7S RhoA (Hirata et al. 1992).

Besides regulating the contraction of sm ooth m uscle cells RhoA also regulates contractility in neurites. The RhoA agonist LPA prom otes grow th cone collapse in a RhoA dependent m anner in neuroblastom a NlE-115 cells. Inactivation of Rho proteins by C3 toxin prom otes neurite outgrow th in a Cdc42 and Racl dependent m anner (Gallo and Letorneau 1998). Studies on prim ary neurons are less clear; in embryonic chick dorsal root ganglions C3 induces axonal outgrow th as in NlE-115 cells. How ever in cultured cortical neurons C3 toxins decreases