Laboratório de Bioquímica e Fisiologia de Microrganismos, Núcleo de Pesquisas em Ciências Biológicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Campus do Morro do Cruzeiro 35.400-000 Ouro Preto, MG, Brazil1
Author for correspondence: R. L. Brandão. Tel: +55 31 3559 1723. Fax: +55 31 3559 1680. e-mail: rlbrand{at}cpd.ufop.br
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ABSTRACT |
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Keywords: signal transduction, nutrient signalling
Abbreviations: MAP, mitogen-activated protein; PKC, protein kinase C
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INTRODUCTION |
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At the post-transcriptional level two different mechanisms seem to be involved: direct phosphorylation of the enzyme (Chang & Slayman, 1991 ; Eraso & Portillo, 1994
) and/or proteolytic degradation of a putative inhibiting protein of the activation process (de la Fuente et al., 1997
).
In spite of the fact that different putative phosphorylation sites present in this enzyme have been described in some detail (Eraso & Portillo, 1994 ), the identity of the protein kinase involved in ATPase phosphorylation is still not known. It was demonstrated that the RAS-cAMP-protein kinase A pathway is not involved in glucose-induced activation of ATPase (Becher dos Passos et al., 1992
). On the other hand, based on the existence in the carboxyl terminus of the plasma membrane ATPase of phosphorylation consensus recognition sites for casein kinase I and calmodulin-dependent protein kinase I, these enzymes were proposed to be involved in this regulation process (Kolarov et al., 1988
; Kemp & Pearson, 1990
; Estrada et al., 1996
). Recently, it was demonstrated that protein kinases involved in the regulation of other plasma membrane proteins would be related to the glucose-induced activation of the plasma membrane H+-ATPase (Goossens et al., 2000
).
Furthermore, by working with different putative inhibitors for protein kinase C (PKC), calmodulin-dependent protein kinase and phosphatidylinositol turnover, we proposed that a phosphatidylinositol type signalling pathway could be involved in the glucose-induced activation of the plasma membrane ATPase (Brandão et al., 1994 ). Indeed, the involvement of phospholipids found in the plasma membrane in the control of ATPase activity had been described by Patton & Lester (1992)
. However, in spite of the fact that glucose stimulates phosphatidylinositol turnover in yeast (Francescotti et al., 1990
), the identification of this hypothetical signalling pathway has not yet been achieved. Nevertheless, Coccetti et al. (1998)
demonstrated the involvement of a phospholipase C in glucose-induced phosphatidylinositol turnover and in the activation of the plasma membrane ATPase of S. cerevisiae. Thus, we originally imagined that the glucose-induced activation of the plasma membrane ATPase could be related to the PKC activation in a similar pathway in yeast cells (Brandão et al., 1994
; Coccetti et al., 1998
).
In fact, the PKC found in yeast and mammalian cells show similar enzymic properties, except for the absence of activation of yeast PKC by tumour-promoting phorbol esters (Ogita et al., 1990 ). Remarkably, by using bovine myelin basic protein (MBP) as a model substrate, it was observed that both enzymes show different specificities. The mammalian PKC phosphorylates several seryl residues, whereas yeast PKC phosphorylates threonyl residues. Moreover, the seryl residues phosphorylated by the mammalian PKC are followed by basic amino acid residues at the carboxyl terminus, but none of the yeast threonyl residues is followed by a basic amino acid residue (Iwai et al., 1992
). In this sense, Eraso & Portillo (1994)
demonstrated that glucose-stimulated phosphorylation of Thr-912 in the ATPase carboxyl terminus is essential to get an increase in the Vmax. Moreover, in the plasma membrane ATPase, this phosphorylation site is not followed by basic amino acid residues.
Since glucose seems to trigger a transduction signal mechanism leading to the activation of the plasma membrane ATPase, one could expect a complete system acting in this process formed by specific glucose receptors, G protein(s), internal signals and the phosphorylating and activating enzyme(s). In this paper we demonstrate that the glucose-induced activation of the plasma membrane ATPase requires the presence of Snf3p (a glucose sensor), the Gpa2 protein (a G protein) and we confirm the involvement of PKC. We also show that phosphorylation of glucose is indeed the internal signal necessary to this activation process and that this signal is transduced via Gpa2p.
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METHODS |
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Measurement of H+-ATPase activity
. For time-course measurements of ATPase activity, cells were incubated at a density of 75 mg ml-1 (wet wt) in a reciprocating water bath shaker at 30 °C. Incubation was carried out in 100 mM MES/Tris buffer (pH 6·5) for 10 min before addition of glucose to a final concentration of 100 mM. At different times, samples containing 375 mg cells (wet wt) were taken from the suspension and the cells collected as quickly as possible on glass fibre filters by vacuum filtration. The cells were quickly removed from the filters and immediately frozen in liquid nitrogen and stored until use.
The procedures used to obtain plasma membranes and to determine ATPase activity were described by Becher dos Passos et al. (1992). Protein was determined according to the Lowry method.
Reproducibility of results.
The experiments were performed at least three times with consistent results. Standard deviations are indicated in each figure.
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RESULTS AND DISCUSSION |
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However, as can be seen in Fig. 1, deletion of the Gpr1p receptor does not decrease markedly the H+-ATPase activation. We also measured the glucose-induced H+ efflux: in the wild-type strain the H+-pumping rate was not statistically different (P>0·05) from a gpr1
mutant [138±30 µmol H+ h-1 (g cell)-1 and 111±15 µmol H+ h-1 (g cell)-1, respectively]. Nevertheless, our results point to the participation of G protein in this activation process since deletion of GPA2 decreased by half the glucose activation of the H+-ATPase (Fig. 2
). In this case the glucose-induced H+-pumping rate was reduced from 192±47·9 µmol H+ h-1 (g cell)-1 in the wild-type strain to 95±37·2 µmol H+ h-1 (g cell)-1 in the gpa2
mutant (values statistically different: P<0·05). These results seem to suggest that the combined receptortransducer system, Grp1p/Gpa2p, does not mediate ATPase regulation.
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Surprisingly, as demonstrated in Fig. 3, only Snf3p seems to be required for a normal glucose-induced activation of the ATPase. Considering that we used 100 mM (±1·8%) glucose in all experiments, it might have been expected that the low-affinity glucose sensor, Rgt2p, would be the sensor. However, there is evidence that Snf3p can contribute to high-level glucose signalling, even taking into account that its own expression is repressed fivefold by high levels of glucose. In an rgt2
mutant the expression of HXT1 is only reduced about fivefold and it is completely absent in the snf3
rgt2
mutant (Ozcan & Johnston, 1995
; Ozcan et al., 1996a
).
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The nature of the internal signalling
Moreover, by using strains with deletions in all genes encoding functional glucose carriers (genes HXT1, 2, 3, 4, 5, 6, 7), and glucose or maltose as signalling molecules, we demonstrated that sugar transport is essential for the regulation of the H+-ATPase activation process. The glucose-induced efflux of protons was reduced from 243±32·5 µmol H+ h-1 (g cell)-1 in the wild-type strain to 5±0·1 µmol H+ h-1 (g cell)-1 in the hxt1-7 mutant (values statistically different: P<0·05). On the other hand, by working with an hxt1-7
gpa2
strain, and using maltose as signalling molecule, we additionally demonstrated the importance of the G protein Gpa2p in the activation process of the plasma membrane H+-ATPase (Fig. 4
). In the wild-type and in the hxt1-7
strains the maltose-induced efflux of protons was not statistically different: 28±7·6 and 34+12·9 µmol H+ h-1 (g cell)-1, respectively (P>0·05). However, in the hxt1-7
gpa2
strain, the maltose-induced efflux of protons was almost zero. Since, in order to be metabolized, maltose should be hydrolysed and phosphorylated, these results seem to suggest that sugar phosphorylation is the internal signal for ATPase activation.
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Due to the fact that in a previous study we demonstrated the involvement of PKC in the glucose-regulated activation of the plasma membrane ATPase (Brandão et al., 1994 ), we decided to clarify this situation by using strains with mutations in the PKC MAP kinase pathway found in yeast cells. In the present study we confirmed the involvement only of PKC1 and not the other MAP kinases of the pathway in H+-ATPase activation (Fig. 6a
, b
). These data confirm that there is a bifurcation after PKC and one of its alternative targets seems to be the plasma membrane ATPase.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 December 2000;
revised 30 April 2001;
accepted 15 June 2001.
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