Sphingoid intermediates, including sphingoid bases, sphingoid base phosphates, and ceramides (), play important roles in regulation of cell growth, differentiation, senescence, and apoptosis (Hannun and Obeid, 2008
; Dickson, 2010
; Nikolova-Karakashian and Rozenova, 2010
). Serine palmitoyltransferase (SPT) mediates the rate-limiting first step in sphingolipid biosynthesis (). Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood (Cowart and Hannun, 2007
). A recent study revealed that yeast Orm proteins, encoded by ORM1
, form a conserved complex with SPT and that their phosphorylation status affects sphingolipid production (Breslow et al., 2010
; ). The authors proposed that sphingolipid levels feedback regulate Orm protein phosphorylation, thus mediating sphingolipid homeostasis (Breslow et al., 2010
). However, several important questions related to this model need answers. For example, whether and which sphingolipid species affect Orm phosphorylation are not known. In addition, how temporal regulation of Orm phosphorylation relates to dynamic changes in sphingolipid biosynthesis is not known. Answers to these questions are required to better understand Orm-mediated sphingolipid homeostasis.
FIGURE 1: Schematic diagram of yeast sphingolipid biosynthesis from endogenous and exogenous precursors. Myriocin is a potent inhibitor of SPT (Sun et al., 2000 ), which is the first and rate-limiting enzyme of the sphingolipid biosynthesis pathway (Buede et al. (more ...)
Another important question concerns how Orm-mediated sphingolipid homeostasis may function in a physiological context. Increasing evidence suggests that various stimuli trigger accumulation of sphingoid intermediates, which in turn function as bioactive molecules mediating cellular responses (Hannun and Obeid, 2008
; Dickson, 2010
). For instance, heat stress–induced sphingoid intermediates act as signaling molecules to induce cellular responses such as translation initiation of heat shock proteins, gene regulation, and cell cycle arrest (Dickson et al., 1997
; Mao et al., 1999
; Jenkins and Hannun, 2001
; Cowart and Hannun, 2005
; Cowart et al., 2010
; Meier et al., 2006
; Han et al., 2010
). It is striking that several groups demonstrated that heat stress induces sphingolipid biosynthesis in a rapid and transient manner (Dickson et al., 1997
; Jenkins et al., 1997
; Jenkins, 2003
; Wells et al., 1998
; Mao et al., 1999
; Skrzypek et al., 1999
), suggesting that biosynthesis of sphingoid intermediates in response to stresses requires precise temporal regulation. We hypothesize that the dynamic changes in sphingoid intermediate levels upon heat stress may be caused by and/or may lead to changes of Orm phosphorylation. If this is the case, heat stress could serve as a model system to address the mechanism of how Orm phosphoregulation functions in the response of sphingolipid biosynthesis to stress in general.
A recent study showed that yeast protein kinases 1 and 2 (Ypk1/2), the homologues of mammalian serum- and glucocorticoid-inducible kinase (Casamayor et al., 1999
), directly phosphorylate Orm proteins in vitro (Roelants et al., 2011
). This result suggests that Ypk kinase may regulate sphingolipid homeostasis through its phosphorylation of the Orm proteins. However, whether Ypk kinase activity indeed affects sphingolipid production has not been examined. Of interest, the Ypk kinases were previously considered to be downstream of heat-induced sphingolipid base accumulation (Sun et al., 2000
; Friant et al., 2001
; Liu et al., 2005
; Hannun and Obeid, 2008
; Dickson, 2010
). Thus, testing for a possible role for Ypk kinase activity in the sphingolipid biosynthesis response to heat stress is an important goal.
Of importance, the Orm phosphorylation state is set not only by protein kinases, but also by phosphatases. A reasonable prediction is that the phosphatases acting on the Orm proteins are likely to be involved in and/or regulated by sphingolipid levels. Protein phosphatase 2A (PP2A) was identified as an attractive candidate for a ceramide-activated protein phosphatase in yeast (Nickels and Broach, 1996
). Inactivation of the regulatory subunits (such as Cdc55) or the catalytic subunits of PP2A was reported previously to suppress the endocytic defects of a mutant with impaired SPT activity (Friant et al., 2000
), suggesting that PP2A may be involved in sphingolipid production. Because PP2A was suggested previously to be involved in multiple cellular signaling pathways (Jiang, 2006
), the challenge is to identify a specific relationship between Cdc55-PP2A and the Orm proteins for regulation of sphingolipid biosynthesis.
In this article, by addressing the questions just raised, we demonstrate how Orm phosphorylation regulation controls sphingolipid biosynthesis in response to heat stress in a kinetically coupled manner. We identify a signaling pathway by which the conserved Pkh-Ypk signaling cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production upon heat stress through precise regulation of Orm protein phosphorylation. Our study will therefore provide a foundation for future studies of sphingolipid-related responses to other stimuli.