In this report, we describe a previously unidentified regulatory mechanism for MLK/Slpr activity in Drosophila. While the role of Slpr in JNK signaling during dorsal closure has been well studied, there has been little evidence to date that Slpr is required for stress response in vivo. This is the first demonstration that Slpr is modified in response to stress. Specifically, phosphorylation within a conserved PXSP motif of Slpr is enriched upon heat or cold treatment and modulates organismal response to thermostress.
Based on the appearance of two forms of SKLC in lysates prepared from in vitro
and in vivo
sources, and the detection of endogenous Slpr in embryos with a phospho-specific antibody, we conclude that under steady state conditions, basal phosphorylation and dephosphorylation of the PXSP site must be occurring. Upon heat shock, p38 kinases were strongly activated ( and 
) and the prevalence of the phosphorylated form of PXSP was increased in the endogenous protein over three-fold. In addition, the heat shock-induced enrichment of phospho-SKLC was diminished in embryos where p38 activity was compromised in the epidermis by RNAi or dominant negative constructs. Thus, while the coincidence of these observations is consistent with a direct relationship, wherein p38 MAPK phosphorylates serine 512 in the PXSP motif upon stress treatment, in vitro
IP-kinase assays with mammalian p38α (Stronach, unpublished data) do not support that interpretation, and alternative explanations are conceivable. For example, other kinases are activated by p38 MAPKs, including the MAPKAP/MK, MSK, and MNK family members 
, which might be responsible for PXSP phosphorylation. Moreover, the enrichment of phospho-PXSP upon temperature shift might not necessarily be due to increased phosphorylation by an active kinase, but rather, loss of phosphatase activity. Thus, it is interesting to note that in several mammalian tissue culture systems, dual-specificity MAPK phosphatases are heat labile, accounting in part for the accumulation of phosphorylated MAPKs to sustain signaling under stress 
. Whether this mechanism is conserved in Drosophila
has not yet been investigated; however, a recent study revealed that the MAPK phosphatase, Puckered, was phosphorylated by JNK and p38 in response to oxidative stress, though the consequences of this modification are still poorly understood 
. Nevertheless, inactivation of MAPK phosphatases could contribute indirectly to the observed increase in phospho-Slpr. Mechanistically, reduced dephosphorylation of a MAPK that normally provides for the basal level of phosphorylation on PXSP, might secondarily enhance this modification, as observed in vivo
. Also, the observation that phospho-SKLC was not enriched in lysates from UV-treated embryos, in which p38 is expected to be active, argues against a general p38 requirement and perhaps in favor of a temperature-dependent mechanism.
Though the details of how p38 MAPKs are involved in regulating Slpr phosphorylation under stress conditions remain to be elucidated, the requirement for p38 MAPK signaling in Drosophila
heat shock response is clear ( and 
). Our results also define a requirement for JNK signaling in response to heat stress. Indeed, both slpr
mutants are more sensitive to prolonged heat shock than control animals. While this result is not that surprising in light of transcriptional profiling studies demonstrating that targets of both JNK and p38 pathways include genes essential for stress response, such as HSPs 
, it does contrast with experiments demonstrating that HSP-mediated inhibition of JNK signaling is protective to cells providing a mechanism for acquired thermotolerance 
. The nature of the experimental systems, cells versus whole organism, or the duration of stress, minutes versus hours, may account for this potential discrepancy in the role of JNK activity in heat stress response. Our findings here, show that loss of either JNK or p38 signaling pathways under heat stress conditions impairs the necessary response, making animals susceptible to prolonged injurious insult. Although phosphorylated active Bsk/JNK was detected in our lysates even under non-stress conditions, possibly accounting for the basal levels of PXSP phosphorylation, we observed no impact on phospho-SKLC when JNK pathway activity was altered positively or negatively. Nor did we detect phosphorylation of SKLC by JNK1 in vitro
(not shown). Taken together, it seems unlikely that feedback regulation of Slpr by Bsk/JNK could account for the temperature dependent enrichment of PXpSP even though the precedent of mammalian MLK3 phosphorylation by JNK as a positive feedback mechanism has been described 
What then is the physiological purpose of the conserved PXSP site? And does the enrichment of the phosphorylated form upon high or low temperature shock have any consequences in vivo? In developmental processes that require Slpr-dependent JNK signaling, we found that mutants in the PXSP site behaved more or less like wildtype Slpr. When overexpressed, the PXAP and PXEP proteins were competent to upregulate JNK signaling in the embryo and to complement a slpr mutant to viability to variable extents. However, when expressed in animals that were also subjected to heat stress conditions, there was a deficit in the response of animals expressing the non-phosphorylatable form, PXAP, and a measurable protection in animals expressing the phospho-mimetic PXEP protein. Moreover, flies expressing the PXAP form had a shorter median survival value at 41 days relative to controls with 51-day median survival in longevity experiments. So flies having this modification (PXEP), or being able to dynamically regulate it (PXSP), were at an advantage relative to flies expressing a form that could not be modified (PXAP), suggesting that phosphorylation at this site is functional and not just a byproduct of heat shock.
The lower overall recovery of flies expressing PXAP transgenes might indicate that they are generally less fit than other genotypes used here. While this is a concern, the results we obtained using several independent transgenic lines provides evidence against a spurious effect on fitness due to the insertion site interfering with an unknown gene. Moreover, the results that PXAP expression in the embryo had a minimal impact on embryonic lethality or signaling under nonstress conditions, coupled with the evidence that UV radiation does not appear to elicit changes in the phosphorylation status at PXSP, as does heat shock, suggests a distinct, physiological response.
While our data suggest a role for PXSP phosphorylation in thermostress signaling, it remains unclear by what mechanism this modification modulates Slpr activity. To probe the effect on Slpr when PXSP phosphorylation is lost, we performed several biochemical experiments (not shown). Proteolytic analysis of PXAP and SlprWT revealed similar proteolysis patterns with or without heat shock, indicating analogous protein folding between the two forms, thus ruling out the notion that the PXAP protein is grossly misfolded. Consistent with that observation, the unfolded protein response pathway was not induced in embryos overexpressing PXAP under non-stress conditions. Furthermore, we were unable to detect a biochemical interaction between Slpr and HSP70 or HSP90 under normal or stress conditions. Thus, while phosphorylation within the PXSP motif might not be required for proper protein folding, other explanations are credible, including regulation of protein turnover, spatial distribution, or activity. JNK phosphorylation of mammalian MLK3 at the serine-proline site regulates its distribution in the cell to modulate signaling intensity 
. We are currently examining the localization of Slpr transgenic proteins under different conditions to explore this possibility. Alternatively, phosphorylation within this motif may affect binding of substrate or upstream effectors. Tests are ongoing to determine the consequences of PXSP phosphorylation at the protein level.
Another question raised by our results is which tissue requires active JNK or p38 signaling during adult heat stress? Accumulating evidence suggests that the nervous system, in particular insulin producing neurosecretory cells, upregulate JNK signaling during oxidative stress response, which counteracts insulin/IGF signaling allowing an adaptive systemic response 
. Additionally, the JNK pathway is normally active in neuronal development, particularly in the mushroom body 
, and in maintaining neuronal homeostasis in both Drosophila
and mammals 
. We have observed that arm-Gal4
directs transgenic Slpr protein expression weakly in larval imaginal discs and nonneural tissue, but strongly in the larval and adult nervous system, primarily in the mushroom bodies of the brain 
. Intriguingly, this structure has recently been linked to a systemic response of adult flies to heat shock, in animals deficient for the mitochondrial phosphatase PGAM5 
. Moreover, this phosphatase has been shown previously to regulate the activity of ASK1, a MAP3K in the JNK signaling pathway 
. Whether arm-Gal4
directed expression of Slpr transgenes in the insulin-producing cells of the adult brain or in other structures including the mushroom body is responsible for the phenotypic differences that we observe in adult stress response will be a topic for future investigations.
The results presented in this report demonstrate that the JNK pathway, including Slpr, is required for heat shock response. Modification of the PXSP site in Slpr is enriched in a temperature dependent fashion and correlates with the degree of susceptibility to heat stress. We argue that phosphorylation of the PXSP motif is important to sustain JNK signaling in attempt to reestablish homeostasis. The presence of phosphorylated Slpr protein at steady state under nonstress conditions might buffer signaling activity from complete failure in rapidly changing conditions.