MSS4, a Phosphatidylinositol-4-phosphate 5-Kinase Required for Organization of the Actin Cytoskeleton in Saccharomyces cerevisiae*

Sylvane DesrivièresDagger §, Frank T. Cooke, Peter J. Parker, and Michael N. HallDagger parallel

From the Dagger  Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the  Imperial Cancer Research Fund Laboratories, P.O. Box 123, Lincoln's Fields, London WC2A 3PX, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The Saccharomyces cerevisiae protein MSS4 is essential and homologous to mammalian phosphatidylinositol-4-phosphate (PI(4)P) 5-kinases. Here, we demonstrate that MSS4 is a lipid kinase. MSS4 has dual substrate specificity in vitro, converting PI(4)P to PI(4,5)P2 and to a lesser extent PI(3)P to PI(3,4)P2; no activity was detected with PI or PI(5)P as a substrate. Cells overexpressing MSS4 contain an elevated level specifically of PI(4,5)P2, whereas mss4 mutant cells have only approximately 10% of the normal amount of this phosphorylated phosphoinositide. Furthermore, cells lacking MSS4 are unable to form actin cables and to properly localize their actin cytoskeleton during polarized cell growth. Overexpression of RHO2, encoding a Rho-type GTPase involved in regulation of the actin cytoskeleton, restores growth and polarized distribution of actin in an mss4 mutant. These results suggest that MSS4 is the major PI(4)P 5-kinase in yeast and provide a link between phosphoinositide metabolism and organization of the actin cytoskeleton in vivo.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phosphorylated phosphoinositides play a crucial role in the transduction of signals induced by a variety of stimuli, including growth factors, hormones, and neurotransmitters. Signaling by phosphoinositides is conserved from yeast to mammalian cells. Phosphatidylinositol (PI)1 is sequentially phosphorylated by a PI 4-kinase to produce PI(4)P and by a PI(4)P 5-kinase to generate PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C, yielding inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (1). IP3 binds to specific receptors and induces the release of calcium from intracellular stores, whereas diacylglycerol activates protein kinase C. The increase in intracellular calcium and the activation of protein kinase C act in synergy to regulate transcription and to control other cellular events. More recently, it has been shown that PI(4,5)P2 can, at least in mammalian cells, be further phosphorylated by PI-3 kinase to generate PI(3,4,5)P3, a mediator of cell growth and survival (Ref. 2 and references therein). In addition, PI(4,5)P2 can directly regulate the organization of the actin cytoskeleton through specific interactions with actin-associated proteins (3-10).

Several lipid kinases able to generate PI(4,5)P2 have been identified in mammalian cells. They fall into two subfamilies (type I and type II), differing both biochemically and immunologically, that together form a novel family of homologous PI kinases with no homology to PI 3-kinases or classical PI 4-kinases (11-16). Zhang et al. (17) have presented evidence that both the type I and type II enzymes generate PI(4,5)P2 by phosphorylating PI(4)P on the D-5 position of the inositol ring. More recently, Rameh et al. (18) have demonstrated that both types generate PI(4,5)P2, but the type I enzyme is a PI(4)P 5-kinase whereas the type II enzyme is a PI(5)P 4-kinase. Both types of enzymes also seem to catalyze the phosphorylation of PI(3)P (17, 18). Hereafter, we collectively refer to the type I and type II enzymes as PIP kinases.

In the yeast Saccharomyces cerevisiae, two predicted proteins, FAB1 and MSS4, show extensive sequence similarity to the mammalian PIP kinases. The FAB1 gene was identified based on the fab1 mutant phenotype of aberrant chromosome segregation. Further characterization of the fab1 mutant indicated that loss of FAB1 causes defects in vacuole function and morphology and in cell surface integrity (19). The MSS4 gene was first identified as a multicopy suppressor of the temperature-sensitive lethality caused by a mutation in the PI 4-kinase gene STT4 (20). MSS4 was also isolated recently as a multicopy suppressor of the growth defect caused by loss of TOR2, a yeast homologue of PI 3- and 4-kinases (21). Unlike FAB1, MSS4 is essential, suggesting that these two proteins have different cellular functions. Based on their homology with mammalian PIP kinases, FAB1 and MSS4 are assumed to be the yeast counterparts of the mammalian lipid kinases, although kinase activity for FAB1 and MSS4 has yet to be demonstrated.

This study was undertaken to determine whether, as predicted by its primary structure, MSS4 is a PIP kinase and to identify its cellular function(s). We demonstrate that MSS4 accounts for most of the PI(4)P 5-kinase activity in yeast and controls organization of the actin cytoskeleton.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Strains, Plasmids, and Media-- The S. cerevisiae strains used in this study are listed in Table I. All strains were isogenic derivatives of JK9-3d. Escherichia coli strain MH4 is Delta (lac)X74 hsr- hsm+ strA leuB600 galE galK. Plasmids used in this study were as follows. Plasmids pSH22, pSH23, and pSD6 are pSEY18 (2 µ URA3), YCplac111 (CEN LEU2), and YCplac33 (CEN URA3), respectively, carrying MSS4 on a 4.1-kb BamHI-XhoI fragment. pSD2 (pGST-MSS4) is the yeast GST-fusion vector pEG-KT (2 µ URA3 leu2-d) (22) containing the full coding region of MSS4. pRHO2 is pC-186, RHO2 (2 µ URA3) (23). Rich media (YPD) and synthetic minimal media (SD, SRaff/Gly) complemented with the appropriate nutrients for plasmid maintenance were as described (24).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Yeast strains used in this study

Expression and Purification of Recombinant GST-MSS4-- GST-MSS4 was expressed in yeast from the GAL1 promotor. Cells containing pGST-MSS4 (JK9-3da/alpha /pSD2) were grown at 30 °C in SRaff/Gly-URA to early logarithmic phase. Expression of the GST-MSS4 fusion protein was induced by the addition of 2% (final) galactose for 8 h. The cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride), lysed with glass beads in a bead-beater, and centrifuged at 500 × g, 4 °C, for 10 min to remove cell debris. GST-MSS4 was precipitated with glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. After washing five times with lysis buffer and five times with 50 mM Tris-HCl, pH 7.5, the beads were stored at -70 °C until use.

Phosphatidylinositol Kinase Assay-- The lipids used in this assay were PI (Lipid Products), PI(3)P (gift from Dr. G. Painter), PI(4)P (Calbiochem), and PI(5)P (gift from Drs. G. Prestwich and G. Peng). Dried lipids were sonicated into 46 µl of assay buffer (25 mM HEPES, pH 7.4, at 25 °C, 2 mM MgCl, 0.2 mM EDTA, 1 mM EGTA, 5 mM beta -glycerophosphate, 1 mM dithiothreitol, and 120 mM NaCl) to give a final concentration in the assay of 100 µM substrate and 300 µM phosphatidylserine (Lipid Products). GST-MSS4 bound to glutathione beads (4 µl) was added to the assay, and the reactions were started by the addition of 15 µl of assay buffer containing 5 µCi [gamma -32P]ATP and 43 µM ATP. Kinase activities were tested at 30 °C for 30 min. The reactions were terminated by the addition of 243 µl of methanol/chloroform (2:1), and the lipids products were extracted by the addition of 58 µl of 2.4 M HCl and 245 µl of chloroform. The lower phase was washed with 239 µl of upper phase (47:48:3, M HCl/methanol/chloroform, v/v/v), and the lipids were analyzed by TLC on a Whatman LKD5 TLC plate as described by Munnik et al. (25). After autoradiography for 1.5 h at room temperature, the phosphorylated spots were scraped, deacylated, and analyzed by HPLC as described (26), using purified tritiated deacylated PI(3,5)P2 (GroPIns(3,5)P2) as an internal standard.

[3H]Inositol Labeling of S. cerevisiae-- SD100 and SD102 were grown at 24 °C to an A600 of 0.7 in medium containing 50 µCi/ml [3H]inositol (Amersham). The cells were harvested by centrifugation and resuspended in one-fifth of the original volume. After control samples had been removed, the cells were incubated at 37 °C. Aliquots (200 µl) were collected, and the cells were arrested by the addition of 500 µl of methanol. The samples were dried in vacuo, and lipid extraction was performed as described previously (26). Some experiments (Fig. 4A) were done without temperature shift. In this case, the cells were labeled at 30 °C, and lipids were immediately extracted, as described above.

Disruption of MSS4-- The entire open reading frame of the MSS4 gene was deleted in MH272-1da/alpha with a polymerase chain reaction-generated fragment containing the HIS3MX6 module (A. Wach, and P. Philippsen, Biozentrum, Basel, Switzerland) flanked by 45 nucleotides homologous to the region either directly upstream of the start codon or downstream of the stop codon of MSS4. The oligonucleotides used for generation of the polymerase chain reaction disruption cassette were 5'-GACAGTTTGCCCTATATCGCTTTTCCCTATCAATAGTTTCTAACTCGCGCCAGATCTGTTTAGCTTGCC-3' and 5'-AGACTGAGTACATAGTCGATAGGTTATTTACCTGTGCCCTACTCACGAGCTCGTTTAAACTGGATGGCG-3'. The disruption was confirmed by Southern blot analysis.

Isolation of an MSS4 Conditional Allele-- Chemical mutagenesis and identification of mutants were performed as described previously (27). Briefly, the plasmid pSH23 was mutagenized in vitro for various times with 1 M hydroxylamine. The level of mutagenesis was assessed by testing the plasmids for complementation of the leucine auxotrophy of the E. coli strain MH4. Plasmid aliquots in which 5% of the LEU2 genes had been inactivated were used to transform SD2-5a/pSH22 (URA3). Leu+ transformants were replica plated at 24 °C on 5-fluoroorotic acid-containing medium to select for loss of the pSH22 plasmid (carrying the wild-type MSS4). The colonies were afterward plated on YPD at 24 and 37 °C for detection of a temperature-sensitive phenotype. Plasmids conferring a temperature-sensitive growth were isolated and retested on the original host strain SD2-5a/pSH22, using the same procedure. The recessive mss4-2ts allele was isolated from 12,000 transformants. The mss4-2ts allele in the temperature-sensitive strain SD102 was complemented by a plasmid-borne copy of MSS4 (pSD6).

Fluorescence Microscopy-- Cells were grown to early logarithmic phase, fixed in formaldehyde, and stained with TRITC-phalloidin (Sigma) to visualize actin, as described previously (28).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Disruption of MSS4 and Isolation of a Conditional Mutant-- To determine the cellular function of MSS4, we constructed a strain deleted for MSS4. The disruption replaced the entire open reading frame of MSS4 with HIS3. In agreement with a previous report (20), we found that MSS4 is essential for growth in our strain background. When a heterozygous diploid mss4Delta ::HIS3/MSS4 strain (SD1) was sporulated and dissected, only the two His- spores gave rise to colonies on rich medium containing either glucose or galactose as a carbon source (data not shown). Because deletion of MSS4 is lethal, we isolated a temperature-sensitive (ts) mutant to facilitate the analysis of MSS4 function (see "Materials and Methods"). The temperature-sensitive mutant SD102, carrying the mss4-2ts allele, grew like wild type at the permissive temperature (24 or 30 °C) but stopped growing 4 h (corresponding to approximately 3 generations) after shift to the restrictive temperature (37 °C).

Inactivation of MSS4 Causes Loss of Cell Viability-- To further assess the consequences of loss of MSS4, the mss4-2ts strain was examined under the light microscope after incubation at the restrictive temperature. The only striking observation was the appearance of "ghost" cells (see Fig. 1), suggesting a loss of viability of the mutant cells at 37 °C. Indeed, the mutant cells started to die after 2 h at 37 °C, as evidenced by staining with the vital dye methylene blue. After 4 h at 37 °C, ~60% of the mss4-2ts cells stained intensely with methylene blue (data not shown), whereas no appreciable staining was observed for a wild-type control.


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 1.   Actin distribution in wild-type and mss4-2ts cells. Logarithmic cultures of wild-type MSS4 (SD100; A-J) and mss4-2ts (SD102; K-T) cells were grown at 24 °C and then shifted to 37 °C. At the indicated times, aliquots were removed, fixed with formaldehyde, and stained with TRITC-phalloidin. The cells were examined with Nomarski optics (B, D, F, H, J, L, N, P, R, T) and by fluorescence to visualize the actin (A, C, E, G, I, K, M, O, Q, S). Arrowheads show "ghost" mss4-2ts cells.

MSS4 Is Required for Organization of the Actin Cytoskeleton-- MSS4 was identified in our laboratory in a screen for multicopy suppressors of the growth defect of a tor2ts strain (21). TOR2 encodes a putative phosphatidylinositol kinase with two essential functions (29-32). One function is to control translation initiation and G1 progression in response to nutrients (33, 34); the second function is to control the organization of the actin cytoskeleton via a Rho-type GTPase switch (35, 36). Overexpression of MSS4 restores both growth (21) and actin organization (data not shown) of a tor2ts mutant defective in the organization of the actin cytoskeleton.

The above results suggest that MSS4, like TOR2, is also involved in organization of the actin cytoskeleton. To test this, we examined if the lack of MSS4 also caused a defect in the polarized distribution of the actin cytoskeleton. Wild-type (SD100) and mss4-2ts (SD102) cells were grown at 24 °C and then shifted to 37 °C. At time points, aliquots were removed, and the cells were fixed and stained with TRITC-phalloidin to visualize F-actin (Fig. 1). When grown at 24 °C, both wild-type cells and mss4-2ts cells displayed the normal cell cycle-dependent distribution of actin (37, 38), with cortical actin patches concentrated in the bud or septum, and actin cables aligned toward the concentrated patches. A shift from 24 to 37 °C caused, in both strains, a heat-induced disorganization of the actin cytoskeleton. Cables were no longer visible, and actin patches were more randomly distributed after 1 h at 37 °C. However, whereas the heat-induced disorganization of the actin cytoskeleton was transient in wild-type cells, lasting only 1-2 h, this perturbation was irreversible in the mss4-2ts cells and became even more severe after 3 or 4 h at 37 °C. Thus, like TOR2, MSS4 is required for organization of the actin cytoskeleton.

Overexpression of RHO2 Suppresses an mss4 Mutation-- The actin cytoskeleton is regulated by the Rho family of small GTPases (for review, see Ref. 39). Since overexpression of RHO2, encoding a member of this family, restored both organization of the actin and growth in a tor2ts, we asked if overexpression of RHO2 could also suppress an mss4-2ts mutation. The mss4-2ts mutant (SD102) was transformed with the high copy number plasmid pRHO2, and transformants were monitored for growth at 37 °C. As shown in Fig. 2A, overexpression of RHO2 restored growth of the mss4-2ts strain at restrictive temperature. We also examined whether overexpression of RHO2 could suppress the actin organization defect of the mss4-2ts strain. SD102 transformed with pRHO2 or with an empty vector was grown at 24 °C, shifted to 37 °C for 4 h, fixed, and stained with TRITC-phalloidin. Contrary to cells carrying an empty vector, which displayed the actin defect, cells transformed with pRHO2 regained the ability to polarize actin patches and to form actin cables (Fig. 2B). The findings that overexpression of RHO2, encoding an actin-related function, suppressed both the growth defect and the actin defect of an mss4-2ts mutant suggest that the essential function of MSS4 is to control the actin cytoskeleton.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2.   Overexpression of RHO2 suppresses an mss4-2ts mutation. A, overexpression of RHO2 suppresses the growth defect of an mss4-2ts mutant. Wild-type cells (SD100) and mss4-2ts (SD102) cells carrying either an empty vector or pRHO2 were streaked on solid rich medium (YPD) and incubated for 2 days at 30 or 37 °C. B, overexpression of RHO2 suppresses the actin organization defect of mss4-2ts cells. mss4-2ts (SD102) cells carrying an empty vector (A and B) or pRHO2 (C and D) were grown in selective media (SD-URA) at 24 °C to logarithmic phase and then shifted to 37 °C for 4 h. The actin content was visualized as described in Fig. 1.

MSS4 Is a PIP Kinase with Dual Substrate Specificity in Vitro-- MSS4 shares high sequence homology with the PIP kinases and has therefore been classified as a PI(4)P 5-kinase (15). However, MSS4 has yet to be demonstrated to have enzymatic activity. To determine whether MSS4 is indeed a PI(4)P 5-kinase, we expressed and purified MSS4 as a GST-MSS4 fusion protein. The GST-MSS4 hybrid was functional as determined by the ability of the GST-MSS4 gene fusion to complement an MSS4 disruption (data not shown). GST-MSS4 was assayed for PI kinase activity using PI, PI(3)P, PI(4)P, or PI(5)P as substrates. Reaction products were resolved by thin layer chromatography and visualized by autoradiography. As shown in Fig. 3A, purified GST-MSS4 had strong kinase activity on PI(4)P and weak activity on PI(3)P; no activity was detected toward PI or PI(5)P. After deacylation and HPLC analysis, the product of the PI(4)P kinase reaction was identified as PI(4,5)P2, and the product of the PI(3)P reaction was identified as PI(3, 4)P2 (Fig. 3, B and C). Thus, as observed previously for at least the mammalian type I PIP kinase, MSS4 phosphorylates PI(4)P on the D-5 position and PI(3)P on the D-4 position in vitro. These results indicate that MSS4 is a dual substrate-specific PIP kinase in vitro, generating PI(4,5)P2 and PI(3, 4)P2 from PI(4)P and PI(3)P, respectively.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Phosphorylation of PI(3)P and PI(4)P by purified MSS4. A, autoradiograph of in vitro kinase reaction products separated by TLC. The activity of purified GST-MSS4 was assayed using 100 µM of PI (lane 1), PI(3)P (lane 2), PI(4)P (lane 3), or PI(5)P (lane 4) as a substrate. The PIP2 spots in lanes 2 and 3 contained 12,411 cpm and 65,338 cpm, respectively. B and C, HPLC chromatogram of the deacylated products of the kinase reactions (closed circles), with deacylated [3H]PI(3,5)P2 (GroPIns(3,5)P2) as internal standard. B, the deacylated PIP2 product of MSS4, using PI(3)P as substrate is identified as GroPIns(3,4)P2. C, the deacylated PIP2 product of MSS4, using PI(4)P as substrate, is identified as GroPIns(4,5)P2.

MSS4 Is the Major PI(4)P 5-Kinase in S. cerevisiae-- To determine the substrate specificity of MSS4 in vivo, we first tested the effect of MSS4 overexpression on cellular phosphoinositide levels. A wild-type strain (SD100) and a strain containing MSS4 on a multicopy plasmid (SD2-5a/pSH22) were labeled with [3H]inositol, and the labeled lipids were deacylated and analyzed by HPLC. Overexpression of MSS4 induced about a 1.7-fold increase in the level of PI(4,5)P2 compared with a wild-type strain (Fig. 4A). Overexpression of MSS4 had no influence on the cellular levels of PI(3)P, PI(4)P, or PI(3, 5)P2. No PI(3, 4)P2 was detected in lipid extracts from the control strain, but a small amount of PI(3,4)P2 (93 ± 6 cpm versus 13,487 ± 497 cpm for PI(4,5)P2) was detected in the strain overexpressing MSS4. Thus, it seems that although MSS4 has the potential to generate PI(3,4)P2, production of this lipid in vivo is minor. To confirm that the increase in cellular PI(4,5)P2 was due to MSS4, we determined whether the strain containing the temperature-sensitive mss4-2ts allele had lowered levels of PI(4,5)P2. Wild-type (SD100) and mss4-2ts (SD102) cells were labeled with [3H]inositol at 24 °C, washed, and shifted to 37 °C. Aliquots were removed at various times, and the tritiated lipid content was analyzed by HPLC. As shown in Fig. 3B, the temperature-sensitive strain SD102 contained levels of PI and PI(4)P comparable with those of the wild-type strain, at both permissive and nonpermissive temperatures. However, the level of PI(4,5)P2 in the mutant strain was moderately reduced already at permissive temperature and severely reduced at nonpermissive temperature. At permissive temperature, SD102 contained about 35% of the amount of PI(4,5)P2 found in wild-type cells. A shift to the higher temperature induced a transient and mild increase in the level of PI(4,5)P2 in wild-type cells, whereas in the mutant the level of PI(4,5)P2 dropped, reaching (within 1 h) approximately 10% of the level found in the wild type. Thus, MSS4 is a PI(4)P 5-kinase both in vitro and in vivo. Furthermore, MSS4 is the major PI(4)P 5-kinase in S. cerevisiae under normal growth conditions.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   MSS4 controls the cellular level of PI(4,5)P2. A, effects of MSS4 overexpression on cellular phosphoinositides. Wild-type cells (SD100; control) or cells overexpressing MSS4 (SD2-5A/pSH22; MSS4) were labeled at 30 °C with [3H]inositol, and [3H]inositol-containing lipids from the cell extracts were deacylated, separated by HPLC, and quantified. B, effect of loss of function of MSS4 on cellular phosphoinositides. Wild-type cells (SD100; open circles) and cells carrying a temperature-sensitive allele of mss4 (SD102; closed circles) were labeled at 24 °C with [3H]inositol and then shifted to 37 °C. Aliquots were collected at the indicated times; the [3H]inositol-labeled lipids from the cell extracts were deacylated and separated by HPLC, and the radioactivity associated with each lipid was quantified.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We demonstrate that, under normal growth conditions, MSS4 is the major PI(4)P 5-kinase in S. cerevisiae. Furthermore, like mammalian PIP kinases, MSS4 has dual substrate specificity in vitro (17, 18). Comparison of the substrate specificities of MSS4 and mammalian PIP kinases indicates that MSS4 is more closely related to type I than to type II PIP kinases, phosphorylating primarily PI(4)P to generate PI(4,5)P2 and to a lesser extent PI(3)P, yielding PI(3,4)P2. Mammalian type I PIP kinases can, in addition, further phosphorylate PI(3,4)P2 to produce PI(3,4,5)P3 (17). Unlike the studies with mammalian PIP kinases, which demonstrated only the in vitro activities of these enzymes, we examined the activity of MSS4 in vitro and in vivo. We found that MSS4 produces PI(4,5)P2 both in vitro and in vivo. However, we did not detect PI(3,4,5)P3 in yeast lipid extracts under any of our experimental conditions, and PI(3,4)P2 was detected only weakly and only when MSS4 was overexpressed. This may indicate that, although MSS4 can generate PI(3,4)P2 in vitro, the production of this lipid in vivo may not be physiologically relevant. Alternatively, synthesis of PI(3,4)P2 might be tightly regulated and might require a very specific stimulus. For example, the synthesis of the recently discovered lipid PI(3,5)P2 (26, 40) is induced by hyperosmotic shock (26). The physiological significance of PI(3,4)P2 remains to be determined.

Several lines of evidence indicate that the essential function of MSS4 is involved in organization of the actin cytoskeleton. First, overexpression of MSS4 suppresses both the growth defect and the actin defect of a tor2ts mutant that is inviable because of an actin defect (Ref. 21 and data not shown). Second, loss of MSS4 causes a growth arrest that is coincident with a disruption of the actin cytoskeleton. Third, overexpression of RHO2, encoding a Rho-type GTPase involved in organization of the actin cytoskeleton (36), suppresses both the growth defect and the actin defect of an mss4-2ts mutant. Finally, overexpression of a type I PIP kinase in mammalian cells induces massive actin polymerization (41, 42). We could not detect any effect of overexpression of MSS4 from a high copy plasmid on the actin cytoskeleton (data not shown), but under these conditions, the cellular level of PI(4,5)P2 was less than doubled (Fig. 3A). Furthermore, a 65% reduction in the cellular level of PI(4,5)P2, as observed in our mss4-2ts mutant at permissive temperature, had no visible effect on the organization of the actin cytoskeleton. These results may suggest that the intracellular levels of PI(4,5)P2 can fluctuate significantly without a major effect on the actin cytoskeleton. More severe changes in PI(4,5)P2 levels, like the 90% decrease in the mss4-2ts mutant at restrictive temperature, might be necessary to affect organization of actin.

How is MSS4 required for organization of the actin cytoskeleton? PIP(4,5)P2 binds and regulates the function of several actin-binding proteins, including profilin (3), gelsolin (4), gCap39 (5), N-WASP (10), ERM proteins (9), alpha -actinin, and vinculin (7, 8). Thus, a simple model for the role of MSS4 in organization of the actin cytoskeleton is that, by generating PIP(4,5)P2, it directly controls the interactions between actin and the actin-binding proteins. Another possibility is that MSS4 signals to the actin cytoskeleton indirectly, through the Rho-type GTPases (43). Mammalian type I PIP kinases are believed to signal to the actin cytoskeleton as downstream effectors of Rho and Rac GTPases (44-46). Our finding that RHO2 is a multicopy suppressor of the mss4-2ts mutation suggests that MSS4 signals to the actin cytoskeleton as part of a signaling pathway involving Rho-type GTPases. This model is strengthened by the fact that MSS4 is a multicopy suppressor of a tor2 mutation, and TOR2 signals to the actin cytoskeleton via RHO1 and RHO2 (21, 36). Furthermore, MSS4 may be in a pathway with PKC1 (see below), which is a direct downstream effector of RHO1 (47, 48).

The S. cerevisiae protein kinase C (PKC) homologue PKC1 plays a major role in ensuring cell integrity (49, 50) and is activated by heat or hypotonic shock (51, 52). Several lines of evidence suggest that MSS4 and PKC1 are involved in a common signaling pathway. First, the MSS4 gene was originally identified as a multicopy suppressor of a temperature-sensitive mutation in the PI 4-kinase gene STT4, which had previously been implicated in PKC1 signaling (20). Second, both MSS4 and PKC1 were isolated as multicopy suppressors of a tor2 mutation (21). One function of TOR2 is to control organization of the actin cytoskeleton by activating RHO1 (36), and both MSS4 and PKC1 can suppress the loss of this function. Third, like a tor2 mutant, an mss4 mutant fails to properly localize actin during polarized cell growth. pkc1 mutants have not been shown to have defects in actin organization, but cells altered in SLT2/MPK1, a mitogen-activated protein kinase activated by PKC1, exhibit delocalized actin (53). Fourth, we observed that high temperature triggers synthesis of PI(4,5)P2, suggesting that MSS4, like PKC1, is activated by heat shock. How MSS4 may be related to PKC1, RHO1, RHO2, and TOR2 in a pathway(s) remains to be determined.

    ACKNOWLEDGEMENTS

We thank Dr. G. Painter for the gift of PI(3)P, Drs. G. Prestwich and G. Peng for the gift of PI(5)P, T. Schmelzle and T. Beck for comments on the manuscript, and Dr. Y. Ohya for communicating results before publication.

    FOOTNOTES

* This work was supported by grants from the Imperial Cancer Research Fund (to P. J. P. and F. T. C.) and from the Swiss National Science Foundation and the Canton of Basel (to M. N. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a long-term European Molecular Biology Organization fellowship.

parallel To whom correspondence should be addressed. Tel.: 41-61-267-2162; Fax: 41-61-267-2149; E-mail: hall{at}ubaclu.unibas.ch.

1 The abbreviations used are: PI, phosphatidylinositol; PI(4,5)P2, phosphatidylinositol 4,5-biphosphate; PI(3,5)P2, phosphatidylinositol 3,5-biphosphate; IP3, inositol 1,4,5-triphosphate; kb, kilobase(s); GST, glutathione S-transferase; HPLC, high performance liquid chromatography; GroPIns(3,5)P2, deacylated PI(3,5)P2.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205[CrossRef][Medline] [Order article via Infotrieve]
  2. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
  3. Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474[Medline] [Order article via Infotrieve]
  4. Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364[CrossRef][Medline] [Order article via Infotrieve]
  5. Yu, F. X., Johnston, P. A., Sudhof, T. C., and Yin, H. L. (1990) Science 250, 1413-1415[Medline] [Order article via Infotrieve]
  6. Janmey, P. A., Lamb, J., Allen, P. G., and Matsudaira, P. T. (1992) J. Biol. Chem. 267, 11818-11823[Abstract/Free Full Text]
  7. Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152[CrossRef][Medline] [Order article via Infotrieve]
  8. Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522[Abstract/Free Full Text]
  9. Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T., Takai, Y., Tsukita, S., and Tsukita, S. (1996) J. Cell. Biol. 135, 37-51[Abstract]
  10. Miki, H., Miura, K., and Takenawa, T. (1996) EMBO J. 15, 5326-5335[Abstract]
  11. Divecha, N., Truong, O., Hsuan, J. J., Hinchliffe, K. A., and Irvine, R. F. (1995) Biochem. J. 309, 715-719[Medline] [Order article via Infotrieve]
  12. Jenkins, G. H., Fisette, P. L., and Anderson, R. A. (1994) J. Biol. Chem. 269, 11547-11554[Abstract/Free Full Text]
  13. Bazenet, C. E., Ruano, A. R., Brockman, J. L., and Anderson, R. A. (1990) J. Biol. Chem. 265, 18012-18022[Abstract/Free Full Text]
  14. Loijens, J. C., and Anderson, R. A. (1996) J. Biol. Chem. 271, 32937-32943[Abstract/Free Full Text]
  15. Boronenkov, I. V., and Anderson, R. A. (1995) J. Biol. Chem. 270, 2881-2884[Abstract/Free Full Text]
  16. Loijens, J. C., Boronenkov, I. V., Parker, G. J., and Anderson, R. A. (1996) Adv. Enzyme Regul. 36, 115-140[CrossRef][Medline] [Order article via Infotrieve]
  17. Zhang, X., Loijens, J. C., Boronenkov, I. V., Parker, G. J., Norris, F. A., Chen, J., Thum, O., Prestwich, G. D., Majerus, P. W., and Anderson, R. A. (1997) J. Biol. Chem. 272, 17756-17761[Abstract/Free Full Text]
  18. Rameh, L. E., Tolias, K. F., Duckworth, B. C., and Cantley, L. C. (1997) Nature 390, 192-196[CrossRef][Medline] [Order article via Infotrieve]
  19. Yamamoto, A., DeWald, D. B., Boronenkov, I. V., Anderson, R. A., Emr, S. D., and Koshland, D. (1995) Mol. Biol. Cell. 6, 525-539[Abstract]
  20. Yoshida, S., Ohya, Y., Nakano, A., and Anraku, Y. (1994) Mol. Gen. Genet. 242, 631-640[Medline] [Order article via Infotrieve]
  21. Helliwell, S. B., Howald, I., Barbet, N., and Hall, M. N. (1998) Genetics 148, 99-112[Abstract/Free Full Text]
  22. Mitchell, D. A., Marshall, T. K., and Deschenes, R. J. (1993) Yeast 9, 715-722[Medline] [Order article via Infotrieve]
  23. Madaule, P., Axel, R., and Myers, A. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 779-783[Abstract]
  24. Guthrie, C., and Fink, G. R. (eds) (1991) Guide to Yeast Genetics and Molecular Biology, Vol. 194, Academic Press, Inc., San Diego, CA
  25. Munnik, T., de Vrije, T., Irvine, R. F., and Musgrave, A. (1996) J. Biol. Chem. 271, 15708-15715[Abstract/Free Full Text]
  26. Dove, S. K., Cooke, F. T., Douglas, M. R., Sayers, L. G., Parker, P. J., and Michell, R. H. (1997) Nature 390, 187-192[CrossRef][Medline] [Order article via Infotrieve]
  27. Sikorski, R. S., and Boeke, J. D. (1991) in Guide to Yeast Genetics and Molecular Biology (Guthrie, C., and Fink, G., eds), Vol. 194, pp. 302-318, Academic Press, Inc., San Diego, CA
  28. Benedetti, H., Raths, S., Crausaz, F., and Riezman, H. (1994) Mol. Biol. Cell 5, 1023-1037[Abstract]
  29. Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. (1993) Cell 73, 585-596[Medline] [Order article via Infotrieve]
  30. Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R., and Hall, M. N. (1994) Mol. Biol. Cell 5, 105-118[Abstract]
  31. Zheng, X. F., Florentino, D., Chen, J., Crabtree, G. R., and Schreiber, S. L. (1995) Cell 82, 121-130[Medline] [Order article via Infotrieve]
  32. Thomas, G., and Hall, M. N. (1997) Curr. Opin. Cell Biol 9, 782-787[CrossRef][Medline] [Order article via Infotrieve]
  33. Barbet, N., Schneider, U., Helliwell, S. B., Stansfield, I., Tuite, M., and Hall, M. (1996) Mol. Biol. Cell 7, 25-42[Abstract]
  34. Di Como, C. J., and Arndt, K. T. (1996) Genes Dev. 10, 1904-1916[Abstract]
  35. Schmidt, A., Kunz, J., and Hall, M. N. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13780-13785[Abstract/Free Full Text]
  36. Schmidt, A., Bickle, M., Beck, T., and Hall, M. N. (1997) Cell 88, 531-542[Medline] [Order article via Infotrieve]
  37. Kilmartin, J. V., and Adams, A. E. (1984) J. Cell Biol. 98, 922-933[Abstract]
  38. Adams, A. E., and Pringle, J. R. (1984) J. Cell Biol. 98, 934-945[Abstract]
  39. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
  40. Whiteford, C. A., Brearley, C. A., and Ulug, E. T. (1997) Biochem. J. 323, 597-601[Medline] [Order article via Infotrieve]
  41. Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., and Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581[Abstract/Free Full Text]
  42. Sakisaka, T., Itoh, T., Miura, K., and Takenawa, T. (1997) Mol. Cell. Biol. 17, 3841-3849[Abstract]
  43. Schmidt, A., and Hall, M. N. (1998) Ann. Rev. Cell Dev. Biol., in press
  44. Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513[Medline] [Order article via Infotrieve]
  45. Hartwig, J. H., Bokoch, G. M., Carpenter, C. L., Janmey, P. A., Taylor, L. A., Toker, A., and Stossel, T. P. (1995) Cell 82, 643-653[Medline] [Order article via Infotrieve]
  46. Van Aelst, L., and D'Souza-Schorey, C. (1997) Genes Dev. 11, 2295-2322[Free Full Text]
  47. Nonaka, H., Tanaka, K., Hirano, H., Fujiwara, T., Kohno, H., Umikawa, M., Mino, A., and Takai, Y. (1995) EMBO J. 14, 931-5938
  48. Kamada, Y., Qadota, H., Python, C. P., Anraku, Y., Ohya, Y., and Levin, D. E. (1996) J. Biol. Chem. 271, 9193-9196[Abstract/Free Full Text]
  49. Levin, D. E., and Bartlett-Heubusch, E. (1992) J. Cell Biol. 116, 1221-1229[Abstract]
  50. Paravicini, G., Cooper, M., Friedli, L., Smith, D. J., Carpentier, J. L., Klig, L. S., and Payton, M. A. (1992) Mol. Cell. Biol. 12, 4896-4905[Abstract]
  51. Davenport, K. R., Sohaskey, M., Kamada, Y., Levin, D. E., and Gustin, M. C. (1995) J. Biol. Chem. 270, 30157-30161[Abstract/Free Full Text]
  52. Kamada, Y., Jung, U. S., Piotrowski, J., and Levin, D. E. (1995) Genes Dev. 9, 1559-1571[Abstract]
  53. Mazzoni, C., Zarov, P., Rambourg, A., and Mann, C. (1993) J. Cell. Biol. 123, 1821-1833[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.