Pmk1 activation in response to glucose deprivation
We have previously described that glucose exhaustion is one of the multiple physiological insults which activate the Pmk1 MAPK signaling pathway in fission yeast [17]. As shown in Figure 1A, removal of glucose by shifting the cells from a rich medium to a similar medium containing glycerol induced a progressive and clear increase in Pmk1 phosphorylation in control cells, reaching its maximum around 90 min, and slowly decreasing thereafter. This alternative carbon source cannot be assimilated unless a minimal amount of glucose is present, and its initial concentration was selected to prevent differential osmotic changes. Virtually the same pattern of activation was observed when the cells were switched to a growth medium employing both glycerol and ethanol as carbon sources (not shown). Interestingly, transfer of exponentially growing cells from rich glucose medium (7% w/v) to osmotically equilibrated medium with glucose concentrations of either 1% or 0.5% did not elicit a significant increase in Pmk1 phosphorylation (Figure 1A), suggesting that full activation of the MAPK cell integrity pathway in S. pombe only takes place after complete depletion of this carbon source.
In fission yeast glucose deprivation triggers a moderate endogenous oxidative stress which is followed by the induced expression of genes like gpx1+ (glutathione peroxidase) and ctt1+ (cytoplasmic catalase). These products play a critical role in the removal of intracellular hydrogen peroxide arising in the change from fermentative to respiratory metabolism [12]. However, preincubation of cell cultures in the presence of N-acetyl cysteine (NAC), a known scavenger for peroxide radicals, did not affect Pmk1 activation in response to glucose withdrawal (Figure 1B). Identical results were obtained when employing other antioxidants like glutathione or alpha-tocopherol (not shown). Hence, Pmk1 activation in the absence of glucose appears due to the lack of this particular carbon source, and unrelated to endogenous oxidative stress.
A novel mechanism is responsible for Pmk1 activation in response to glucose deprivation
We next tried to identify the signaling elements involved in the activation of the Pmk1 MAP kinase module in response to glucose exhaustion. Rho2, one of the six Rho GTPases found in S. pombe proteome, is a main positive regulator upstream of the cell integrity pathway in some stress conditions [18, 19]. Importantly, Rho2-dependent regulation of Pmk1 activity is mediated through Pck2, one of the two orthologs of protein kinase C (PKC) present in this organism [8, 18, 19], while Pck1, the second PKC ortholog, appears to negatively regulate basal MAPK activity by an unknown mechanism [18]. The essential GTPase Rho1 has been also proposed to function as positive regulator of Pmk1 activity [20]. Although we had previously described a partial defect in Pmk1 phosphorylation in rho2Δ cells after 90 min in the absence of glucose [18], repeated exhaustive analysis of this mutant under the above conditions showed that maximal MAPK phosphorylation was actually very similar to that of control cells, except for a delay in the activation kinetics at earlier times (Figure 2A). Therefore, this new evidence suggests that the role of Rho2 during signal transduction to the Pmk1 cascade in response to glucose exhaustion is, at most, rather modest.
Notably, MAPK activation was strongly compromised in a mutant lacking Pck2 and slightly affected in Pck1-less cells, whereas simultaneous deletion of rho2+ in either pck2Δ or pck1Δ cells did not significantly alter the activation response shown by the single mutants (Figure 2A). These results suggest that Pck2 is the key element involved in full signal transmission of glucose deprivation to the Pmk1 cascade. Moreover, as compared to the Rho2-deleted strain, Pmk1 activation in the absence of glucose remained virtually unaffected in control or rho2Δ cells expressing a dominant negative version of Rho1 (T20N) (Figures 2B and 2C), which constitutively binds to GDP and behaves like a lack of function version of this GTPase [23, 24]. Therefore, neither Rho2 nor Rho1 appear to be major determinants in Pck2-dependend signaling to the Pmk1 MAPK cascade in response to glucose exhaustion.
Rho5 GTPase functions in a redundant fashion to Rho1 and plays a nonessential role during stationary phase and in the process of spore wall formation [25]. It is worth to mention that Rho5 levels are almost undetectable in exponentially growing cells, but increase significantly under glucose starvation [25], thus making this GTPase a potential candidate to modulate Pmk1 activation in a Pck2-dependent fashion. However, as compared to control cells, the enhanced Pmk1 phosphorylation induced by glucose depletion was neither affected by rho5+ deletion nor modified in rho5Δ rho2Δ double mutant cells (Figure 2D). Moreover, simultaneous deletion of rho5+ did not aggravate the defective Pmk1 activation observed in pck2Δ cells (Figure 2E). Notably, Pmk1 activation was still observed in glucose-depleted cells of a rho5Δ mutant expressing a dominant negative allele of Rho1 (T20N) (Figure 2F). This finding rules out the possibility that both GTPases functionally replace each other during signal transduction to the MAPK module. We also observed a clear Pmk1 activation after glucose exhaustion in rho2Δ cells expressing a dominant negative allele of Cdc42 (T17N), which is an essential GTPase involved in the regulation of cell morphogenesis in fission yeast (Figure 2G) [26]. As a whole, the above evidences indicate that Rho1, Rho2, Rho5, or Cdc42 GTPases have not significant role in Pmk1 activation in response to glucose limitation, suggesting the existence of unknown additional element/s to activate the cell integrity MAPK cascade via Pck2 under this condition.
Functional Glucose/cAMP pathway is required for full Pmk1 activation in response to glucose deprivation
In fission yeast the Glucose/cAMP signaling pathway is involved in the regulation of multiple cellular events, including sexual differentiation, spore germination, osmotic stress response and glucose sensing [14, 27]. The main members of this pathway are the G-protein coupled receptor Git3, a heterotrimeric G protein composed of the Gpa2 Gα, the Git5 Gβ, and the Git11 Gγ subunits, plus adenylate cyclase Cyr1, and the cAMP-dependent protein kinase, which in turn is composed by regulatory (Cgs1) and catalytic (Pka1) subunits. In the presence of glucose, Gpa2 Gα subunit binds GTP and activates Cyr1, promoting an increase in cAMP levels which activate Pka1 [27]. Pka1 phosphorylates and negatively regulates the activity of Rst2, a transcription factor responsible for the induced expression of genes like fbp1+, encoding fructose-1,6-bisphosphatase, whose activity is critical for gluconeogenesis and adaptation to grow on non-fermentable carbon sources (i.e, in the absence of glucose) [14]. Considering such precedents, we analyzed the possible effect of the Glucose/cAMP pathway in Pmk1 activation during glucose deprivation. In comparison to control cells, glucose removal resulted in an important decrease in Pmk1 activation in strains deleted in Git3, Gpa2, or Pka1 (Figure 3). On the contrary, Pmk1 activation remained unaffected in rst2Δ cells (Figure 3). These findings suggest that under glucose limitation an operative cAMP pathway is necessary for full activation of the Pmk1 signaling cascade, and that this control is independent on Rst2 function.
Pmk1 activation in response to glucose deprivation requires de novo protein synthesis
To gain further insight into the mechanisms responsible for Pmk1 activation during glucose limitation we analyzed this response in mutant cells of the fission yeast lacking MAPK Sty1, the core element of the SAPK pathway [8]. As shown in Figure 4A, both basal Pmk1 phosphorylation and activation increased in the sty1Δ mutant as compared to control cells after glucose withdrawal. This was a result rather expected because of the previous demonstration that Sty1 negatively regulates Pmk1 phosphorylation through the induced expression of several MAPK phosphatases (Pyp1, Pyp2, and/or Ptc1) able to downregulate both Sty1 and Pmk1 in vivo[21]. Surprisingly, we observed that pre-treatment of growing cultures of wild type cells with cycloheximide, a protein synthesis inhibitor, fully suppressed Pmk1 activation during glucose exhaustion (Figure 4B, upper panel). Moreover, this response appears to be specific since a strong Pmk1 activation was observed in cycloheximide-treated and untreated cells under saline stress (Figure 4B, lower panel). These results strongly support that in fission yeast the stress by glucose limitation signals to the cell integrity pathway through a hitherto unknown mechanism which requires de novo protein synthesis.
Pmk1 reinforces fission yeast adaptive response to metabolic stress imposed by glucose limitation
To explore the biological significance of Pmk1 role during glucose deprivation we first determined whether the absence of this MAPK might affect cell viability during growth adaptation from a glucose-based medium to another with a non-fermentable carbon source. In this context, it has been described that the SAPK pathway and its effector Sty1 are critical in fission yeast to allow adaptation from fermentative to respiratory metabolism [12, 13]. This is confirmed by results in Figure 5A, indicating that, contrary to wild type cells, the growth of sty1Δ cells was impaired when transferred from YES medium to a similar medium in which 7% glucose was substituted by 2% glycerol plus 3% ethanol. The shift to a medium containing 3% glycerol plus 0.05% glucose yielded the same results (not shown). Notably, either pmk1Δ cells or a mutant strain expressing a catalytically dead version of the MAPK Pmk1 displayed a growth defect in respiratory medium that was not observed in the presence of glucose (Figure 5A). This defect did not alleviate by the addition of NAC to the culture medium (Figure 5A), suggesting that endogenous oxidative stress was not the cause underlying this phenotype. As a whole, the above evidence supports the idea that Pmk1 activity contributes to cell growth during the adaptation of fission yeast from fermentative to respiratory metabolism.
An attractive possibility about how the cell integrity pathway might favour fission yeast growth during respiration would be that Pmk1 activity positively affects the expression of fructose-1,6-bisphosphatase (fbp1+), whose activity is critical to achieve growth in the absence of glucose [28]. Confirming this prediction, Northern blot experiments showed that the strong increase in fbp1+ expression during growth in a non-fermentable carbon source was decreased and delayed in pmk1Δ cells as compared to control cells (Figure 5B). Since fbp1+ transcriptional activation is positively regulated by the Sty1 pathway through Atf1 transcription factor [13], we also analyzed the effect of Pmk1 absence in the levels of Pyp2, a tyrosine phosphatase which dephosphorylates both Sty1 and Pmk1, and whose expression is dependent on the Sty1-Atf1 branch [8, 29]. Again, the increased expression of both pyp2+ m-RNA and Pyp2 protein levels was markedly delayed in pmk1Δ cells as compared to control cells after glucose removal (Figures 5B and 5C), thus supporting the conclusion that Pmk1 stimulates the expression of Sty1-Atf1 dependent genes during transition from fermentative to respiratory metabolism.
Because the stress-induced expression of fbp1+ and pyp2+ genes is positively regulated by Sty1 via Atf1, we considered the possibility that the delayed expression of both genes in pmk1Δ cells during the shift to a non-fermentable carbon source might result from an altered kinetics in the activation of the SAPK pathway. Therefore, we comparatively analyzed Sty1 phosphorylation during glucose deprivation in control versus pmk1Δ cells. As shown in Figure 5D, glucose withdrawal induced a quick activation of Sty1 in control cells that was maintained and slowly decreased after 3-4 hours in the presence of non-fermentable carbon sources. However, the kinetics of Sty1 activation in pmk1Δ cells was clearly altered, with a more pronounced dephosphorylation after the initial activation, and the activation maintained for longer times (Figure 5D). Similarly, despite a decreased mobility shift and expression observed early after transfer from fermentative to respiratory medium, Atf1 protein levels (expressed as a genomic copy of the atf1+ gene tagged with two copies of the HA epitope and six histidine residues) remained high in pmk1Δ cells at longer incubation times as compared to control cells (Figure 5E). Notably, the late activation of both Sty1 and Atf1 prompted in the absence of Pmk1 is in good agreement with the delayed expression pattern observed for Fbp1 or Pyp2 (Figures 5B and C). Taken together, these results suggest that in fission yeast Pmk1 positively regulates the timely activation of the SAPK pathway during the switch from fermentative to respiratory metabolism.