Hyperosmotic Stress Induces the Rapid Phosphorylation of a Soybean Phosphatidylinositol Transfer Protein Homolog through Activation of the Protein Kinases SPK1 and SPK2

Abstract

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress. INTRODUCTIONPhosphatidylinositol transfer proteins (PITPs) were identified originally by their ability to serve as diffusible carriers of phosphatidylinositol (PtdIns) and to a lesser extent phosphatidylcholine (PtdCho) from one distinct membrane compartment to another by using an in vitro assay (Wirtz, 1991). In recent years, several intriguing and critical biological roles beyond the transfer of phospholipids have been attributed to yeast and animal PITPs. The yeast PITP (Sec14p) is an essential protein that is required for cells to properly execute the formation of secretory vesicles from the Golgi complex (Bankaitis et al., 1990). A considerable body of evidence suggests that Sec14p serves as a "molecular sensor" to monitor and regulate the levels of PtdIns, PtdCho, and potentially diacylglycerol in the Golgi complex of yeast (Skinner et al., 1995;Kearns et al., 1997). In addition, Sec14p has been implicated in modulating the activity of a PtdIns 4-kinase that regulates protein secretion (Hama et al., 1999).An essential role for PITPs also is observed in mammals and Drosophila, in which the loss of PITP function leads to specific neurodegenerative diseases (Hamilton et al., 1997;Milligan et al., 1997). At the cellular level, the mammalian PITP is known to be required for inositol lipid signaling, secretory vesicle formation from the trans -Golgi network, and the fusion of secretory vesicles to the plasma membrane (Hay and Martin, 1993;Cunningham et al., 1995;Kauffmann-Zeh et al., 1995). Although yeast and animal PITPs are very similar...

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro

“…Northern blot analysis showed that OsPI4K1 is constitutively expressed in various tissues, which may indicate an essential role of OsPI4K1 in rice growth and development. Previous studies have shown that the phosphatidylinositol signaling pathway is involved in many developmental processes [12,13]. Hormone treatment and various environmental factors including salt and drought stress, as well as a hypersensitive reaction (HR) can stimulate the metabolism of phosphoinositides [42,43] showed that salicylic acid probably acts through the phosphoinositide signal transduction system in cucumber.…”

“…Up to now, many of the components involved in PI signaling in animal cells have been identified but relatively few in plant cells [9]. In plants, besides their roles in calcium signalling, phosphoinositides have been implicated in a num-ber of environmental stress signaling processes, including responses to osmotic stress, light stress, pathogen attack, and in regulating plant leaf and flower development [10][11][12][13]. PI4P and PI (4,5) P 2 are known to affect the activity of several enzymes, including the plasma membrane H + -ATPase [14] and phospholipase D [15] and can interact with cytoskeletal components [16].…”

Molecular Responses to Osmotic Stresses in Soybean

“…Osmotic stress (NaCl or sorbitol) can induce the expression of Ssh1p and the phosphorylation of Ssh1p proteins (Kearns et al, 1998), suggesting its role in the phospholipids signalling of osmoprotective responses. Further studies revealed that two soybean serine/threonine protein kinases, SPK1 and SPK2, are responsive to hyperosmotic stress by phosphorylating Ssh1p (Monks et al, 2001). Immunoprecipitation assays demonstrated that the two protein kinases do not belong to MAP kinases, indicating that Ssh1p is not part of the well-known osmotic stress-induced MAPK pathway.…”

Sucrose non-fermenting 1-related protein kinase 2 (SnRK2): a family of protein kinases involved in hyperosmotic stress signaling

“…Mutating Ser-158 and Thr-159 to aspartic acid in the activation loop of OSRK1 or deleting its C-terminal 73 amino acids significantly reduced the enzyme activity (Chae et al, 2007). Monks et al (2001) showed that when overexpressed in yeast, two soybean SnRK2s (SPK1 and SPK2) were activated by high hyperosmolarity. A Vicia faba AAPK homolog has been shown to mediate stomatal closure in guard cell protoplast, in an ABA dependent pathway (Li et al, 1996;.…”

“…SPK3 is induced by exogenously supplied ABA while SPK4 remains unaffected. Two other members of SnRK2s subfamily, SPK1 and SPK2, from soybean were shown to be activated by hyperosmotic stress (Monks et al, 2001). Kobayashi et al (2004) identified 10 SnRk2 protein kinases from rice (Oryza sativa) genome and expressed them in cultured cell protoplasts.…”