The related PH domain-containing proteins Slm1 and Slm2 were originally identified as effectors of the PI4,5P2
and TORC2 signaling pathways and shown to be essential for growth and actin polarization (3
). The studies presented here extend these earlier observations and demonstrate that Slm proteins are also targets of sphingolipid-dependent signaling. We show that Slm1 is essential for growth and proper actin polarization under conditions where de novo sphingolipid synthesis is compromised, suggesting that Slm proteins and sphingolipids cooperate to promote cell survival. Our results further establish a role for sphingolipid signaling pathways in modulating Slm function during heat stress. One role of sphingolipids may be to indirectly modulate Slm activity by regulating PI4,5P2
synthesis required for Slm1 and Slm2 plasma membrane targeting (34
). However, a second, essential role of the sphingolipid-activated signaling pathway is to stimulate Slm phosphorylation on both serine and threonine residues and this phosphorylation event is vital for survival under heat stress conditions.
Sphingolipids signaling is specifically induced in response to heat stress in yeast and the kinetics of Slm phosphorylation correlate well with heat-induced changes in sphingoid base metabolites. Heat stress induces a rapid increase in the sphingoid bases PHS and DHS. This increase is transient; levels peak after 10 to 15 min, returning to basal levels within 60 min (19
). PHS and DHS are rapidly metabolized to PHS-1P and DHS-1P, which peak 15 min after heat stress (21
) followed after approximately 1 h by an increase in ceramide metabolites via de novo synthesis (58
). Thus, PHS or a PHS metabolite may promote Slm phosphorylation by activating an upstream kinase. Consistent with such an idea, we found that Slm phosphorylation levels are significantly diminished in a yeast mutant defective for the sphingoid base-activated kinases Pkh1 and Pkh2, suggesting that the Pkh signaling cascade regulates Slm function. Slm1 and Slm2 lack canonical consensus Pkh/PDK1 phosphorylation motifs, which are present in all known Pkh targets (9
). In agreement with this, Pkh1 failed to directly phosphorylate Slm proteins in vitro. Thus, Slm1 and Slm2 phosphorylation involves an as-yet-unidentified kinase whose activity is modulated by the Pkh signaling cascade. Pkh kinases are known to directly activate several kinases, including Pkc1p, Sch9, and the related kinases Ypk1 and Ypk2. Our data thus far exclude Ypk1/2, and it remains to be determined which kinase(s) directly phosphorylates Slm1/2.
Heat-induced Slm1 phosphorylation is, at least in part, counteracted by the Ca2+/calmodulin-dependent protein phosphatase calcineurin. We show that Slm1 and Slm2 both physically interact with Cna1 via a calcineurin docking site present in their C termini and that deletion of this calcineurin-binding site in Slm1 or treatment of yeast cells with the calcineurin inhibitor FK506 increases basal levels of Slm phosphorylation on both serine and threonine residues. Interestingly, calcineurin activation in an early phase of the heat shock response may also be mediated by sphingolipids (PHS-1P) through stimulation of Ca2+ influx. Thus, sphingolipids may modulate Slm phosphorylation both positively and negatively during heat stress. PHS-1P may trigger the calcineurin-dependent dephosphorylation of Slm observed immediately following heat shock, whereas PHS stimulates Pkh-dependent rephosphorylation of Slm proteins during the recovery period. Together, our data suggest that Slm activity is modulated via a phosphorylation/dephosphorylation cycle with Pkh kinases and calcineurin antagonistically regulating Slm activity.
How does phosphorylation/dephosphorylation affect Slm function? The finding that the specific calcineurin inhibitor FK506 in combination with PHS can partially suppress the growth defect of the temperature-sensitive slm1-ts slm2Δ mutant at the nonpermissive temperature argues for a negative role of calcineurin in Slm regulation and a positive role of sphingolipids in enhancing Slm activity. Consistent with a negative role of calcineurin in Slm regulation, deletion of the calcineurin-binding site does not abolish Slm function. Accordingly, the Slm1ΔCN mutant not only is functional and suppresses growth and actin defects of slm1-ts slm2Δ mutant cells at 38°C (data not shown) but also is more effective than wild-type SLM1 in restoring proper actin polarization in pkh mutant cells.
Our data further demonstrate that phospho-Slm plays a critical role during heat stress and is required for maintaining cell survival and proper actin polarization. This conclusion is supported by biochemical and genetic data showing that conditions that enhance Slm phosphorylation (treatment of cells with FK506 and PHS) also suppress the lethality and actin defects associated with loss of Slm function. Furthermore, identification and functional characterization of one heat-induced phosphorylation site in Slm1, Ser659, using mass spectroscopic analysis and mutagenesis studies directly implicates phosphorylation in survival during heat stress. Replacement of Ser659 in Slm1 with Ala, which prevents phosphorylation, affects Slm1 function under heat shock conditions only. Accordingly, while this Slm1 variant can complement lethality of the slm1-ts slm2Δ mutant under physiological growth conditions (30°C), it fails to do so at 38°C. Thus, phosphorylation may increase Slm1 activity or promote interaction with a cellular component necessary for maintaining growth and proper actin polarization under environmental stress conditions. In agreement with such a hypothesis, the Slm1Ser659D mutant appears to be hyperactive in vivo compared to the wild-type protein. Due to the close proximity of Ser659 to the calcineurin binding site (amino acids 668 to 682), it is tempting to speculate that Ser659 is the site dephosphorylated by calcineurin. Further studies, however, are needed to determine the identity of the residues dephosphorylated by calcineurin and its relation to Ser659.
Sphingolipid-dependent activation of the Pkh pathway is essential for growth, and activated Pkh kinases modulate at least two essential signaling branches required for cell wall synthesis and actin polarization. Our genetic and biochemical studies suggest that Slm proteins function as effectors in one Pkh-regulated signaling branch or act in a parallel, functionally redundant pathway. Perhaps more in favor of the former, we found that an increased dosage of SLM1 can partially rescue the growth and actin cytoskeleton organization defects of the temperature-sensitive pkh1-ts pkh2Δ cells when these cells are osmotically stabilized. Conversely, an increased dosage of PKH1 suppressed neither the growth nor actin defects of the slm1-ts slm2Δ mutant at nonpermissive temperature (data not shown). In agreement with the notion that the Pkh signaling cascade stimulates Slm activity, mutations that enhance or mimic phosphorylation (Slm1Ser659D and Slm1ΔCN) confer an enhanced ability to suppress the growth and actin defects of pkh1-ts pkh2Δ mutant cells. Together, our data are most compatible with a model in which Slm proteins function in a branch of the Pkh signaling pathway to promote actin polarization during heat stress.
Finally, our studies suggest that Slm proteins are required for aspects of sphingolipid-dependent transport processes, because delivery of the arginine permease Can1 to the plasma membrane, which is dependent on de novo sphingolipid synthesis, is blocked in cells lacking Slm activity. In yeast, ongoing sphingolipid biosynthesis is required for the formation of lipid raft domains in the ER and may play a role in the fusion of COPII vesicles with cis
-Golgi membranes (51
). The defect in ER exit of Can1 in slm-ts
mutants would thus be consistent with a common role of Slm proteins and sphingolipids in protein delivery to lipid raft microdomains. Interestingly, plasma membrane delivery of the lipid raft-associated protein Pma1 appeared not to be significantly affected in slm1-ts slm2
Δ mutants. The reason for the intracellular retention of Can1 only is currently unknown, but it may be due to the preferential association of Can1 and Pma1 with raft domains of different lipid compositions along the secretory pathway (45
) and at the plasma membrane (39
). Can1 is thought to associate with raft domains in the ER (45
), whereas Pma1 raft association has been proposed to occur predominantly in the Golgi apparatus (4
). Thus, Can1 and Pma1 may be transported via different subpopulations of vesicles that are differentially sensitive to loss of Slm function. In support of such a hypothesis, immunofluorescence studies revealed more significant overlap of Slm1 signal with Can1 and Sur7, compared to Pma1 (Fig. S1). Thus, Slm proteins may play a role in the trafficking of Can1/Sur7-containing vesicles and their targeting to specific membrane domains at the plasma membrane. Additional studies will need to address this issue.
Slm1 and Slm2 were previously implicated in vesicular trafficking to the cell surface, and several proteins, including the Rab-type GTPase Sec4 and the v-SNARE Snc1, necessary for fusion of secretory vesicles with the plasma membrane, were mislocalized in slm1-ts slm2
Δ mutants (3
). While the precise role of Slm proteins in the exocytotic pathway is not known, there is precedence for a functional connection between Snc1 and sphingolipid biosynthesis in this process. Sphingoid bases and ceramides can modulate secretion and endocytic recycling functions of the redundant v-SNAREs Snc1 and Snc2, because addition of exogenous PHS to the growth medium can restore Golgi-to-plasma membrane transport in yeast mutants defective for Snc1/2 and the t-SNAREs Sso1/2 (41
). In addition, the growth defect of the snc1
Δ mutant can also be suppressed by altering the availability of different ceramide precursors (14
). How alterations in sphingolipids suppress the sorting defects of the snc1
Δ mutant is unclear, but it could be due to changes in sphingolipid signaling or membrane curvature and fluidity. Perhaps in support of a signaling mechanism, we found that the Can1 transport defect is phenocopied by a yeast mutant defective in Pkh function, suggesting that Pkh kinases and Slm proteins affect Can1 trafficking by a common mechanism. The role of Slm proteins and Pkh1/2 kinases in Can1 plasma membrane delivery may be dependent on their roles in regulating actin cytoskeletal polarization, because intracellular retention of Can1 is also observed upon disruption of actin filaments by latrunculin A. While the precise mechanism by which Slm proteins affects Can1 transport remains to be determined, our studies suggest that Slm proteins act downstream of a sphingolipid-derived signal to control the organization of the actin cytoskeleton in response to heat stress. Thus, Slm proteins are subject to control by multiple signaling pathways, including PI4,5P2
TorC2 and sphingolipid-activated Pkh kinases, and it will be interesting to determine whether and how input of these different signals is integrated to temporally and spatially affect actin organization.