Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways

Abstract

<jats:p>Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55–protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.</jats:p>

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

“…This phosphorylation occurs in a graded fashion that may finely tune Orm activity in response to sphingolipid levels. Similarly, Orm1 activity is adjusted in response to manipulation of Orm2 expression levels, with increased Orm2 expression causing a corresponding increase in Orm1 phosphorylation and vice versa (Liu et al 2012;Sun et al 2012). The mechanism by which phosphorylation blocks Orm1/2-mediated inhibition of SPT requires further investigation, but one important feature is that phosphorylation triggers a change in the higher order assembly of the SPOTS complex from a more oligomeric state to a more monomeric state (Fig.…”

“…Specifically, mutations that block Orm protein phosphorylation reduce steady-state sphingolipid levels and prevent up-regulation of SPTactivity when sphingolipid synthesis is disrupted. These mutations therefore cause a pronounced hypersensitivity to inhibitors of sphingolipid synthesis ); conversely, phosphomimetic mutations confer resistance to these drugs (Sun et al 2012). The physiologic importance of Orm proteins in sphingolipid metabolism is further underscored by the finding that the acute increase in sphingolipid synthesis in response to heat stress is also mediated by Orm1/2 phosphorylation (Sun et al 2012).…”

“…These mutations therefore cause a pronounced hypersensitivity to inhibitors of sphingolipid synthesis ); conversely, phosphomimetic mutations confer resistance to these drugs (Sun et al 2012). The physiologic importance of Orm proteins in sphingolipid metabolism is further underscored by the finding that the acute increase in sphingolipid synthesis in response to heat stress is also mediated by Orm1/2 phosphorylation (Sun et al 2012). Lastly, several groups have recently examined how changes in yeast sphingolipid levels are translated into changes in Orm activity (see section below on mechanisms for sensing sphingolipids).…”

“…Furthermore, the increased phosphorylation of Orm1/2 caused by inhibition of sphingolipid synthesis is paralleled by a corresponding increase in Ypk1 activity (Roelants et al 2011). This stimulation of Ypk1 activity is in turn the result of Torc2-mediated phosphorylation of Ypk1 at Thr662 (Roelants et al 2011;Niles et al 2012;Sun et al 2012).…”

17q21 locus and ORMDL3: an increased risk for childhood asthma

“…The regulation of sphingolipid metabolism is incompletely understood and likely complex through multiple interconnected mechanisms (37). Although de novo sphingolipid synthesis may only play a minor contribution to the total cellular sphingolipid pool (46), homeostatic regulators of de novo synthesis such as ORMDL stabilize cellular sphingolipid levels in the face of external perturbations (37,38,47). Previous studies linking asthma to sphingolipids have been centered on inflammatory and allergic mechanisms related to the sphingolipid mediator S1P (48)(49)(50)(51)(52)(53).…”