Ras2 and Ras1 Protein Phosphorylation in Saccharomyces cerevisiae*

(Received for publication, February 4, 1997, and in revised form, May 14, 1997)

Jennifer L. Whistler Dagger and Jasper Rine §

From the Division of Genetics, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

This work describes the phosphorylation of Saccharomyces cerevisiae Ras proteins and explores the physiological role of the phosphorylation of Ras2 protein. Proteins expressed from activated alleles of RAS were less stable and less phosphorylated than proteins from cells expressing wild-type alleles of RAS. This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras protein per se. In addition, phosphorylation of Ras protein was not dependent on proper localization of the Ras2 protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p. The preferred phosphorylation site on Ras2 protein was identified as serine 214. This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 protein on nearby serine residues. A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.


INTRODUCTION

The RAS genes were discovered as the transforming factors of acutely oncogenic retroviruses and were later recognized as virally transduced forms of cellular proto-oncogenes. Cellular RAS genes are activated by mutation in a wide variety of human cancers, providing the first example of a common molecular oncogenic mechanism for cancer (1-5).

Ras genes encode small guanine nucleotide-binding proteins and are highly conserved among eukaryotes. Mammalian cells have at least three distinct Ras proteins, Ha-Ras, N-Ras, and Ki-Ras (6), all of which can act as oncogenes when activated by certain mutations. The Ras superfamily of proteins function as molecular switches that regulate cell growth and differentiation. Ras protein is in its active form when bound to GTP and in its inactive form when bound to GDP. In this way, the ratio of GTP to GDP-bound forms of Ras protein determines the amount of Ras-mediated signaling.

The proper function of Ras protein requires cycling between the GTP and GDP-bound states in response to a nutrient or differentiation signal. All Ras proteins have intrinsic GTPase activity, and the regulation of the ratio of GTP to GDP-bound forms of the protein is modulated partially through regulation of this activity by proteins that stimulate the GTPase activity (7). In fact, mutant forms of Ras protein that fail to hydrolyze GTP produce unregulated cell growth or inappropriate differentiation.

Signal transduction pathways involving Ras proteins are responsible for the developmental fates of several different tissues in metazoans (8-11). Studies of Ras in these signal transduction cascades have led to a unified model for metazoan Ras signaling through a mitogen-activated kinase cascade. Saccharomyces cerevisiae contains two RAS genes, RAS1 and RAS2 (12), that both modulate growth. In contrast to all metazoans studied to date, the effector for RAS in S. cerevisiae is adenylyl cyclase, rather than a member of the mitogen-activated kinase pathway (13). Thus, although the function of RAS is conserved from S. cerevisiae to metazoans, the effectors for these Ras proteins have diverged.

In all species examined, Ras proteins are subject to several post-translational modifications at their carboxyl termini. These carboxyl-terminal modifications are conserved among Ras proteins from metazoans to yeast and are dependent on the presence of a carboxyl-terminal prenylation motif. The modifications include farnesylation of the most carboxyl-terminal cysteine (14), followed by endoproteolytic removal of the three carboxyl-terminal residues (15), and subsequent methylation of the carboxyl group on the newly generated carboxyl-terminal prenyl-cysteine (16). There may also be a reversible palmitoylation of the cysteine residue adjacent to the prenyl-cysteine (17). These modifications play a significant role in the proper localization of Ras protein to the plasma membrane.

The activity of some alleles of Ha-Ras is modulated by phosphorylation (18). Some naturally occurring activated mutant Ha-RasVal12 proteins have a second mutation that changes an alanine to a threonine at position 59 (A59T). Phosphorylation occurs on these Ha-RasVal12-Thr59 mutant proteins on threonine 59. This A59T mutation has been detected only in viral Ras oncoproteins that also contain the G12V mutation as well, and may arise as a suppressor of G12V. Indeed, Ha-RasVal12-Thr59 protein transforms fibroblasts less efficiently than does Ha-RasVal12 protein (18).

The two yeast Ras proteins, Ras1p and Ras2p, are also phosphorylated (19). However these phosphorylations have not been characterized with respect to their position in the protein or their biological function. Because phosphorylation frequently plays a regulatory role in biology, and because the regulation of Ras proteins in yeast is still poorly understood, we characterized the phosphorylation of yeast Ras protein to determine what role(s) it might play.


EXPERIMENTAL PROCEDURES

Media and Genetic and Molecular Methods

Yeast strains were grown in casamino acid medium lacking uracil and/or tryptophan (CAA), yeast minimal medium (YM), or rich medium (YPD) (Difco) containing 2% glucose (Sigma). 5-FOA was used at a concentration of 1 g/liter to select against URA3 function. Diploids were sporulated on minimal sporulation medium (20). Solid media contained 2% agar (Difco). Yeast cells were transformed by the lithium acetate method (21). Doubling times of yeast strains were determined by measuring the A600 of logarithmically growing cultures at 30 °C. For analysis of growth of phosphorylation mutants, all drugs or compounds were added to the YPD media after autoclaving.

All cloning and other molecular methods were performed according to standard methods (22) unless otherwise specified. DNA sequencing was performed on double-stranded templates by the di-deoxy chain termination method, either using the Sequenase 2.0 kit (U. S. Biochemical Corp.) on double-stranded denatured templates with 35S-dATP or by using cycle sequencing with fluorescent nucleotides on double-stranded templates and an ABI automated sequencer.

PCR1 Reactions

All PCR products for cloning were made with Vent DNA polymerase (New England Biolabs) at 1 unit per 100-µl reaction. The reactions were in buffer containing 10 mM KCl, 20 mM Tris, pH 8.8, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 250 µM each dNTP, 1 µM each primer, and 1-10 ng of template DNA. Denaturing temperature was 94 °C. Annealing temperature was 2-5 °C below the Tm of the primer with the lowest Tm. Extension was at 72 °C for 1 min for each kilobase of DNA amplified. Amplification was for 35 cycles.

To sew PCR products together, 2 µl of each template product were added to the standard buffer above with no oligo primers and an addition of 0.1 mM tetramethylammonium chloride. Five cycles were then run in this buffer in the absence of primers to generate full-length product template; the flanking primers were then added at 1 µM concentration, and 35 additional cycles were done to amplify the full-length product.

Oligo-directed Mutagenesis

Oligonucleotide-directed mutagenesis was performed as described previously (23) using single-stranded DNA generated from RAS2 plasmid pJR1039 and RAS2Val19 plasmid pJR1040.

Heat Shock Sensitivity Tests

Analysis of heat shock sensitivity was performed by growing strains for 2 days at 30 °C into stationary phase (A600 4-5) in liquid casamino acid medium lacking tryptophan. Cells were diluted 10 × in distilled H2O and subjected to a 90-s heat shock at 55 °C in a thermocycler (MJ Research Inc.). Cells were then further diluted in distilled H2O and plated onto solid casamino acid medium lacking tryptophan. Percentage survival from heat shock was calculated by comparing the number of colonies arising from heat-shocked cells to the number obtained from cells from the same initial dilution that had not been heat shocked.

Strain Construction

Cells deleted for CDC25 and carrying a CEN TRP1-marked CDC25 plasmid (pCDC25-TRP1) were transformed with a high copy URA3-marked plasmid carrying TPK1 (pJR1857). The resulting strain RAS1 RAS2 cdc25Delta pCDC25-TRP1 pTPK1-URA3 (JRY5416) was grown in medium containing tryptophan to allow loss of the CDC25-TRP1 plasmid. These cells were then replica-plated to medium lacking tryptophan to identify colonies that had lost the CDC25-TRP1 plasmid, one of which became strain RAS1 RAS2 cdc25Delta pTPK1-URA3 (JRY5417), which is genotypically cdc25Delta but otherwise isogenic to JRY5416.

The ras1Delta ras2Delta pTPK1 strain (JRY5404) was constructed in several steps, JRY3001 MATalpha ade2 his3 leu2 trp1 ura3 can1 (W303 background) was transformed with a PCR product designed to disrupt the RAS1 open reading frame. The PCR product was synthesized using Vent DNA polymerase using oligo primers JW040 and JW041. Primer JW040 5'-CAGGTAAACAAAATTTTCCCTTTTTAGAACCATATGGTACTGAGAGTGCACC-3' contained 30 bp complementary to the non-coding strand of RAS1 just upstream of the ATG start codon and generated a NdeI site (underlined) utilizing the ATG start codon. The remaining 16 bp (in bold type) were complementary to the region of the pRS vectors (24) flanking the insertion of the selectable marker genes for these vectors. Primer JW041 5'-CAAAACCATGTCATATCAAGAGAGCAGGATCGCGGTATTTCACACCGC-3' contained 31 bp complementary to the coding strand of RAS1 just downstream of the stop codon and 16 bp (in bold type) complementary to the vector flanking the insertion of the selectable marker genes for these vectors. This primer pair was used to amplify the HIS3 gene from vector pRS303. The resulting PCR product contained the entire HIS3 gene flanked by 30 and 31 bp of RAS1 sequence 5' and 3' of the HIS3 gene, respectively, to target recombination of HIS3 into the RAS1 locus. The PCR product was used to transform JRY3001, and His+ transformants were selected. In approximately half the His+ transformants, the RAS1 gene was replaced by HIS3, as confirmed by DNA blot hybridization (data not shown). The resulting strain, MATalpha ade2 his3 leu2 trp1 ura3 can1 ras1Delta ::HIS3, was JRY5412. This strain was the parent of the bcy1Delta strain described below (JRY5410). The ras1Delta ::HIS3 strain was transformed with the TPK1-URA3 high copy plasmid pJR1857 (JRY5427) and then deleted for RAS2 using a ras2Delta ::LEU2 disruption plasmid (pJR1858). In this ras2::LEU2 the LEU2 gene replaced the RAS2 coding region from the ClaI site 560 bp upstream of the ATG start codon to the MscI site 636 bp into the open reading frame. Disruption was confirmed by the inability of resulting LEU2 transformants to grow on solid medium containing 5-FOA, which selects against the pTPK1-URA3 plasmid, which is required for viability in the ras1Delta ras2Delta strain. Approximately half the resulting LEU2 transformants were unable to grow on 5-FOA. One of these was designated JRY5404 and had the genotype MATalpha ade2 his3 leu2 trp1 ura3 can1 ras1Delta ::HIS3 ras2Delta ::LEU2 pTPK1-URA3.

The bcy1Delta strain (JRY5410) was constructed using the ras1Delta ::HIS3 strain described above (JRY5412) using a linear fragment from plasmid pJR1071 that contained the BCY1 gene disrupted by the LEU2 gene. The disruption was confirmed by testing heat shock sensitivity of the LEU2 transformants (data not shown).

The ras1Delta ::HIS3 strains (JRY5407 and JRY5408) were constructed in two steps to create an unmarked deletion (25). The RAS1 disruption plasmid (pJR1877) lacked the 270-bp HpaI-MscI fragment internal to RAS1. A partial digest of this plasmid with HindIII, which cut within the remaining RAS1 sequence, created a linear fragment that directed insertion to the RAS1 locus (JRY5428). The resulting URA3 transformants were streaked on 5-FOA medium to select for a recombination event that removed the vector with the URA3 gene, leaving behind an unmarked partial deletion allele of RAS1 (JRY5407). The disruption allele was confirmed by DNA blot hybridization. The strain with the ras1 disruption allele was transformed with a URA3 RAS2Val19 plasmid (pJR1861) (JRY5408).

Creation of Isogenic Set of Integrated RAS2 Strains with Various RAS2 Alleles

The phenotypes of various RAS2 alleles were evaluated after integrating these alleles in a strain lacking the wild-type RAS1 and RAS2 alleles (JRY5404). To construct TRP1-integrating versions of the desired RAS2 allele the XhoI-BamHI fragment containing the RAS2 alleles from various plasmids was subcloned into TRP1-marked integrating vector YIplac204, which had been digested with both SalI and BamHI. The resulting plasmids were then digested with NdeI and re-ligated to remove the small polylinker-containing region, making EcoRV a unique site within the TRP1 gene. Linearization of these plasmids with EcoRV directs integration into the TRP1 locus.

Plasmid Constructions

The high copy TRP1-marked RAS2 plasmid (pJR1244) was constructed by cloning the 1.9-kb EcoRI-SalI fragment containing the entire RAS2 gene from pJR827 into pRS424. The TRP1-marked CEN RAS2 plasmid (pJR1051) was constructed by cloning the 1.9-kb EcoRI-SalI fragment into pRS314. The TRP1-marked CEN RAS2Val19 plasmid (pJR1052) was constructed by cloning the 1.9-kb EcoRI-SalI fragment containing the entire RAS2Val19 gene from pJR828 into pRS314. Plasmid pJR827 was constructed by cloning the 1.9-kb ClaI-HindIII RAS2 gene into pRS306. Plasmid pJR828 was constructed by cloning the 1.9-kb ClaI-HindIII fragment containing the entire RAS2Val19 gene into pRS306. Plasmid pJR1039 was constructed by cloning the 1.9-kb EcoRI-SalI RAS2 fragment from pJR827 into pRS316. Plasmid pJR1040 was constructed by cloning the 1.9-kb EcoRI-SalI RAS2Val19 fragment from pJR828 into pRS316. The CEN TRP1-marked RAS2 plasmid (pJR1878) was generated by cloning a XhoI-BamHI fragment containing the entire RAS2 gene from pJR1039 into pRS414.

The high copy URA3-marked TPK1 plasmid (pJR1857) was constructed by cloning a PCR fragment containing the entire TPK1 gene into pRS426. The TPK1 PCR product was amplified using Vent DNA polymerase using oligo primers JW094 and JW095. Primer JW094, 5'-GGAAGCTTGCAAAGCCAGTGTCCCAACG-3' was complementary to the non-coding strand at the 5' end of the TPK1 gene and generated a HindIII site. Primer JW095, 5'-GGAAGCTTGAACGCCGTTCCATCTCCG-3' was complementary to the coding strand at the 3' end of TPK1 and also generated a HindIII site. The resulting PCR product was purified with a Millipore ultrafree-MC microcentrifuge unit, digested with HindIII, gel-purified, and cloned into pRS426, resulting in pJW167, in which the open reading frame of TPK1 was in the same orientation as URA3.

The high copy RAS1 plasmid pJR1859 was constructed by cloning a HindIII fragment containing the entire RAS1 gene into the high copy TRP1 vector YEplac112. In the resulting plasmid (pJR1859) the RAS1 was in the same orientation as TRP1. The high copy RAS1Val19 plasmid (pJR1860) was constructed by cloning a PCR fragment containing the entire RAS1Val19 gene into the high copy TRP1 vector YEplac112. The RAS1Val19 PCR fragment was generated by the PCR sewing method. First, two PCR products were made both of which incorporated the RAS1Val19 mutation into the product by use of a mutagenic oligo primer. One product amplified the RAS1 region 5' of amino acid 19, utilizing oligonucleotides JW134 and T7 as primers. T7 annealed to the polylinker region of RAS1 plasmid pJW99 that was used as the template for the PCR reaction. Primer JW134 5'-GCAGATTTACCAACGCCAACTCCACCGACAACTAC-3' was complementary to the coding strand of RAS1 and introduced the Gly-19 right-arrow Val mutation (bold type, underlined). The other product amplified the RAS1 region 3' of amino acid 19, utilizing oligonucleotide JW133 and reverse as primers. Reverse primer annealed to the polylinker region of RAS1 plasmid pJW99 which was used as the template for the PCR reaction. Primer JW133 5'-GTAGTTGTCGGTGGAGTTGGCGGTGGTAAATCTGC-3' was complementary to the non-coding strand of RAS1 and introduced the Gly-19 right-arrow Val mutation (bold type, underlined). Both products were purified, and 2 µl of each of the previous products were used as template for a PCR sewing reaction (see below) utilizing primers T7 and reverse. The resulting full-length RAS1Val19 PCR product was purified, digested with HindIII, and cloned into the TRP1-marked high copy vector YCplac112 to create pJR1860, in which RAS1Val19 was in the same orientation as the TRP1 gene. The presence of the RAS1Val19 mutation was confirmed by sequencing. The RAS1 plasmid used as template for the PCR reactions described above (pJR1879) was constructed by cloning the HindIII fragment containing RAS1 into the polylinker of pRS425.

A plasmid encoding a fusion protein in which the green fluorescence protein (GFP-S65Y) was joined to the amino terminus of the RAS2 coding region was used to evaluate the localization of Ras2p in living cells. This plasmid was derived from a high copy TRP1-marked vector (pJR1136, a gift from Cindy Trueblood) that contained a HindIII-XbaI fragment with the strong constitutive GPD promoter and the PGK1 terminator. The RAS2 coding region was inserted between the promoter and the terminator by inserting an EcoRI-SalI fragment (from pJR1880) into the EcoRI and SalI sites of pJR1136, forming pJR1881. As a result, RAS2 was under the control of the GPD promoter. In the next step, a very bright variant of the GFP gene (S65Y) (26) was amplified from a plasmid pRH431 (a gift from Randy Hampton) with the oligonucleotide primers JW121 and JW122. 5'-CCGAGCTCATGAGTAAAGGAGAAGAAC-3' (JW121) was complementary to the non-coding strand of vbGFP and introduced a SacI site (underlined) just 5' of the ATG start codon of vbGFP (in bold type), and 5'-CCGAATTCTTTGTATAGTTCATCCATG-3' (JW122) was complementary to the coding strand of vbGFP and introduced an EcoRI site (underlined) just 5' of the stop codon for vbGFP. This EcoRI site allowed a SacI-EcoRI fragment of GFP to be cloned into the GPD-RAS2 plasmid (pJR1881) creating an amino-terminal fusion of GFP to the RAS2 gene (pJR1862). The GFP-ras2-SSIIS plasmid (pJR1863) was constructed by substituting the wild-type RAS2 gene on an EcoRI-SalI fragment from pJR1862 with an EcoRI-SalI PCR fragment containing the ras2-SSIIS allele. The ras2-SSIIS PCR fragment was generated as above from plasmid pEG(KG)RAS2-SSIIS utilizing oligonucleotide primers JW067 and JW127. 5'-CCGAATTCATGCCTTTGAACAAG-3' (JW067) was complementary to the non-coding strand at the 5' end of RAS2, and 5'-CCGTCGACGCGTTTCTACAACTATTTCC-3' (JW127) was complementary to the coding strand of the RAS2 gene prior to the cysteine to serine mutations.

Creation of Phosphopeptide Site Mutant Plasmids

Plasmids marked with the TRP1 gene and containing the serine to alanine mutations at RAS2 positions 198, 202, 207, and 214 (pJR1864, pJR1865, pJR1866, and pJR1867) were created by subcloning the 1.0-kb PstI fragments from pJR1868, pJR1869, pJR1870, and pJR1871, respectively, into the vector fragment of PstI-digested RAS2 plasmid pJR1244 and selecting the correct orientation. Plasmid pJR1868, pJR1869, pJR1870, and pJR1871 were generated by oligo-directed mutagenesis using single-stranded DNA from RAS2Val19 plasmid pJR1040 as template. Oligonucleotide primers JW115, JW116, JW117, and JW118 which were complementary to the coding strand were used to generate pJR1868, pJR1869, pJR1870, and pJR1871, respectively.

Creation of Other Serine to Alanine Mutations by PCR Sewing

PCR products were generated under standard conditions, and sewing products were made as described above using flanking primers JW137 and JW124. These sewing products were transformed into a ras1Delta ras2Delta yeast strain (JRY5404) that carries a URA3-marked RAS2 plasmid, simultaneously with linearized TRP1-marked RAS2-gap plasmid, pJR1882, that was deleted for the HpaI fragment of RAS2, and TRP1 transformants were selected. Strains were then grown on medium containing 5-FOA to select for successful gap repair. Plasmids from these strains were recovered (27) and transformed into Escherichia coli strain DH5alpha . DNA sequencing was used as a final check on the sequence of each mutation created.

The RAS2-gap plasmid, pJR1882, was constructed by digesting RAS2 plasmid pJR1244 with HpaI and re-ligating to remove the 1.5-kb RAS2-containing insert.

Immunoblot Analysis

Samples were harvested at various culture densities, as described, and frozen at -80 °C until processing. Immunoblot analysis was performed essentially as described previously (28), only the rat monoclonal antibody, Y13-259 (Oncogene Sciences), was used at a 1:250 dilution at 4 °C.

Labeling of Cells

Cells were grown to an A600 of approximately 1.0 in medium with 5 µM KH2PO4 and 10 µM (NH4)2SO4 to limit cells for phosphate and sulfate (LPSM). 2 A600 units of cells were pelleted and resuspended in 2 ml of fresh LPSM. 1 mCi of inorganic 32P (Amersham Corp.) or 1 mCi of 35S-trans-label (ICN) was added to cells that were metabolically labeled at 30 °C for 3 h, unless otherwise specified. Labeled cells were then pelleted and washed with 1 ml of YM media with 0.1% NaN3, and the pellets were frozen at -80 °C.

Immunoprecipitation of Ras Proteins Under Native Conditions

Labeled cells were thawed and resuspended on ice in 50 mM Tris, pH 7.5, 20 mM MgCl, 100 mM NaCl, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride (NLB) at 10 OD/ml. Cells were disrupted with glass beads (29). Cell lysates were removed to a clean chilled tube, and glass beads were washed with one additional volume of NLB. Lysate and wash were pooled and centrifuged at full speed in a microcentrifuge at 4 °C for 10 min to pellet cell debris. The supernatants were added to 1.5 × volume of bovine serum albumin-pretreated activated charcoal, vortexed, and pelleted repeatedly until no charcoal residue remained.

Protein G-Sepharose beads conjugated to the anti-Ras antibody Y13-259 (Oncogene Sciences) were prepared using standard procedures (30).

Immunoprecipitations

Equivalent protein for each lysate was added to a 10-µl bed volume (100-µl slurry) of protein G-Sepharose/Y13-259 beads and incubated end over end at 4 °C for 2 h. Lysate was removed, and beads were washed 3 × 100 cv with ice-cold NLB, and then with 3 × 100 cv with ice-cold NLB lacking Nonidet P-40. Immunoprecipitated proteins were eluted from beads in 20 mM Tris, pH 8.0, 20 mM EDTA, 2% SDS, 2 mM GTP, 2 mM GDP at 70 °C for 5 min. Eluate was removed from beads, spun at 4 °C for 5 min to remove any insoluble material, and stored at -20 °C.

Analysis of Immunoprecipitations

Proteins from immunoprecipitations were electrophoretically separated on 12% SDS-polyacrylamide gels, unless otherwise stated. Gels were fixed in 25% isopropyl alcohol, 10% HOAc for 30 min, dried at 60 °C for 1 h, and placed on film at -80 °C and/or exposed to a PhosphorImager cassette (Molecular Dynamics).

Purification of GST-Ras2 Fusion Protein

A yeast strain deleted for RAS1 and RAS2 and carrying the p(EG)KG-RAS2 plasmid (31), which expresses a GST-Ras2 fusion protein from the galactose-inducible Gal1 promoter (JRY5429), was grown in 5 liters of YM with 2% raffinose as a carbon source to an A600 of 0.2 at 30 °C. Galactose was added to 4% and the cells continued to grow overnight at 30 °C for 4 more doublings. Cells were harvested and washed with STMN (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl, 10 mM Tris, pH 7.4) and frozen at -80 °C. Protein was purified as published (31) only no detergent was added. Protein was eluted by digesting the bead-bound protein with thrombin 2 × for 30 min at room temperature. Purification of Ras2p with 12 amino acids from GST was to near-homogeneity from the single affinity chromatographic step. A total of 5 mg of Ras2 protein was obtained from the 5 liters of induced cells. The identity of this protein as Ras was confirmed by immunoblot (data not shown). The resulting Ras2 protein was then further purified by reverse phase high performance liquid chromatography on a C18 column. The protein invariably eluted as two peaks that could not be resolved under the conditions used. A portion of the purified protein was treated with alkaline phosphatase, and the treated protein was eluted as a single peak on high performance liquid chromatography analysis, indicating that the second peak represented phosphorylated Ras2p.

Ras2 peptides were generated by digestion with LysC, AspN, or ArgC (Boehringer Mannheim) according to manufacturer's instructions. Peptides were then individually purified by high performance liquid chromatography over a C18 reverse phase column and subjected to analysis by mass spectrometry.


RESULTS

The Phosphorylation of Ras2 Protein Was a Dynamic Modification

In most cases in which phosphorylation plays a regulatory role on a protein, the half-life of phosphorylation is shorter than the half-life of the protein. Therefore we compared the half-life of the entire Ras2 protein pool and the half-life of phosphorylated Ras2 protein. A strain deleted for ras1 and ras2 and carrying a high copy RAS2 plasmid (JRY5430) was pulse-labeled with either 32Pi or 35S-trans-label and chased into cold medium. The Ras2p was then immunoprecipitated and analyzed by SDS-PAGE. The half-life of the Ras2 protein pool under these conditions was greater than 5 h, whereas the half-life of the phosphorylated pool was less than 1 h (Fig. 1). Based on these data, phosphorylation of Ras2p was dynamic and was therefore potentially a regulatory modification.


Fig. 1. Ras2p phosphorylation is dynamic. A strain deleted for RAS1 and RAS2 (JRY5404) was transformed with a plasmid encoding RAS2. Cells were pulse-labeled with either 32P or 35S for 15 min in LPSM medium and then chased into the same medium lacking label. Samples were removed from the pulse and at 1 and 4 h after chase. Ras2 proteins from these cells were then immunoprecipitated and analyzed by PAGE. Ras2p had a half-life of approximately 5 h, whereas phosphorylated Ras2p had a half-life of less than 1 h.
[View Larger Version of this Image (23K GIF file)]

Activated RAS Alleles Produced Hypophosphorylated Ras Protein

If phosphorylation of Ras2p regulated its activity, then mutations that affect the activity of Ras2p might affect its level of phosphorylation. We therefore tested whether constitutively activated alleles of Ras had an altered level of phosphorylation. Cells with null alleles of chromosomal RAS1 and RAS2 (JRY5404) were transformed with a high copy plasmid carrying either RAS2 (pJR1244) or RAS2Val19(pJR1912), which encodes a Ras2 protein with impaired GTPase activity due to the G19V mutation. This mutation is analogous to the G12V mutation of oncogenic forms of human Ras protein. The resulting yeast strains, ras1Delta ras2Delta pRAS2 (JRY5430) and ras1Delta ras2Delta pRAS2Val19 (JRY5470), were radiolabeled with 32P or grown in the same medium without radiolabel. The Ras proteins from the labeled cells were immunoprecipitated and analyzed for phosphorylation, and the level of Ras protein in the unlabeled cells was analyzed by immunoblot. Cells carrying only RAS2Val19 had 10-fold less steady-state level of Ras protein than cells carrying only RAS2 (data not shown). Thus, to evaluate the effect of the RAS2Val19 allele on phosphorylation of Ras protein per se, the difference in steady-state protein levels was compensated for when loading immunoprecipitates onto the gel. The Ras2 protein was significantly more phosphorylated than Ras2Val19 protein (Fig. 2A). At face value, these data indicated that activated Ras2p was hypophosphorylated relative to wild-type Ras2p.


Fig. 2. Activated RAS alleles produce hypophosphorylated Ras protein. Strains null for RAS were transformed with plasmids encoding RAS2 and RAS2Val19 (A) and RAS1 and RAS1Val19 (B), and the steady-state protein levels of these strains were analyzed by immunoblot. Beads only lane refers to an immunoprecipitation done with protein G-Sepharose beads to which no antibody was cross-linked. Phosphorylation of activated Ras proteins was less than 10% that of wild-type Ras protein. C, a strain deleted for RAS1 and RAS2 (JRY5404) was transformed with a plasmid encoding either RAS2 (JRY5430) or RAS2Val19 (JRY5470). Cells were pulse-labeled with 32P in LPSM medium and then chased into the same medium lacking the radiolabel. Samples were removed at 1, 3, and 5 h after chase. Ras2 proteins from these cells were then immunoprecipitated and analyzed by PAGE. Ras2Val19 protein was less easily phosphorylated and less easily de-phosphorylated than was wild-type Ras2p.
[View Larger Version of this Image (27K GIF file)]

The effect of activating mutations on Ras2 protein steady-state level and phosphorylation state was unprecedented and unexpected. Therefore, the impact of an activating mutation on Ras1 protein phosphorylation was evaluated to determine whether the effect of an activating mutation in RAS2 reflected a general trend for activated alleles of RAS or was an idiosyncrasy of the Ras2 protein. Cells lacking chromosomal RAS1 and RAS2 (JRY5404) were transformed with a high copy plasmid carrying either RAS1 (pJR1859) or RAS1Val19 (pJR1860). The resulting yeast strains, ras1Delta ras2Delta pRAS1 (JRY5405) and ras1Delta ras2Delta pRAS1Val19 (JRY5406), were radiolabeled with 32P or grown in the same medium without radiolabel. As before, the Ras proteins from the labeled cells were immunoprecipitated and analyzed for the extent of phosphorylation, whereas the level of Ras proteins in the unlabeled cells was analyzed by immunoblot. As with Ras2Val19p, cells carrying only RAS1Val19 expressed approximately 10-fold less steady-state level of Ras protein than cells carrying only RAS1 (data not shown). Also, as before, the difference in steady-state protein levels was therefore compensated for when loading the gel to compare the extent of phosphorylation of activated versus non-activated alleles of RAS1. Ras1p was more phosphorylated than Ras1Val19p (Fig. 2B). Thus, RAS1 and RAS2 shared the following two features: activated alleles of either protein resulted in a substantial decrease in steady-state Ras protein level; and even when taking changes in protein level into account, the activated alleles resulted in a substantial decrease in the phosphorylation of both Ras1p and Ras2p.

To determine whether the observed decreased phosphorylation of Ras2Val19 protein was a result of more rapid turnover of the Ras2Val19 protein, we analyzed the turnover rate of the phosphorylated forms of the Ras2Val19 protein compared with the turnover rate of the phosphorylated forms of wild-type Ras2p. A strain deleted for ras1 and ras2 and carrying a high copy RAS2 plasmid (JRY5430) or a high copy RAS2Val19 plasmid (JRY5470) was pulse-labeled with 32Pi and chased into cold medium. The Ras2p was then immunoprecipitated and analyzed by SDS-PAGE. Although the Ras2Val19 protein attained a phosphorylated state more slowly that wild-type Ras2p, the phosphorylated pool of the Ras2Val19 protein was significantly more stable that the phosphorylated Ras2p pool (Fig. 2C), suggesting that rapid turnover of the Ras2Val19 protein was not responsible for its lower steady-state level of phosphorylation. Rather, Ras2Val19 protein was apparently a poorer substrate for the putative Ras2p kinase and/or the Ras2p phosphatase.

Ras Protein Phosphorylation Was Not a Means of Feedback Regulation

The reduction in phosphorylation of Ras2Val19 protein suggested that the phosphorylation of Ras proteins might be tied in some way to the activity of the cAMP-dependent kinase pathway, which is over-stimulated by Ras2Val19p. Because the physiological effects of RAS2Val19 are dominant, it was possible to test whether feedback regulation from the cAMP-dependent protein kinases was responsible for the differential phosphorylation of Ras2 and Ras2Val19 protein. To test whether expression of Ras2Val19 protein could prevent the phosphorylation of wild-type Ras2p in the same cell, two strains with the genotypes ras1Delta RAS2 pRAS2Val19 (JRY5408) and ras1Delta RAS2 (JRY5407) were radiolabeled with 32P and evaluated for Ras protein phosphorylation by immunoprecipitation. The presence of RAS2Val19 did not prevent the phosphorylation of wild-type Ras2 protein in cells with both RAS2 and RAS2Val19 alleles (Fig. 3A, compare lanes 2 and 3). Therefore, reduced phosphorylation of Ras2p was not due to enhanced activity of the cAMP signaling pathway.


Fig. 3. Stimulation of the Ras-cAMP pathway does not alter Ras2p phosphorylation. A, cells co-expressing activating and wild-type alleles of Ras2 protein were radiolabeled with 32P, and the Ras2 proteins from these cells were immunoprecipitated and analyzed by SDS-PAGE. B, cells expressing wild-type Ras2 protein but also containing activating mutations in the Ras-cAMP signaling cascade were radiolabeled with 32P, and the Ras2 proteins from these cells were immunoprecipitated and analyzed by SDS-PAGE. Beads only lane refers to an immunoprecipitation done with protein G-Sepharose beads to which no antibody was cross-linked.
[View Larger Version of this Image (41K GIF file)]

As an independent evaluation of the role of elevated cAMP levels on the phosphorylation state of Ras proteins, other modifications that cause activated phenotypes in yeast cells were tested for whether they reduced the phosphorylation of Ras2p. Overexpression of TPK1, which encodes one of several cAMP-dependent protein kinases, yields activated phenotypes similar to those associated with RAS2Val19 (32). Strains disrupted for BCY1 the regulatory subunit of the cAMP-dependent protein kinases TPK1, TPK2, and TPK3 also have activated phenotypes (32). Strains containing these activating mutations were examined for extent of phosphorylation of the wild-type Ras2 protein. Although the strains ras1Delta ras2Delta pTPK1 pRAS2-2µ (JRY5409) and ras1Delta bcy1Delta pRAS2-2µ (JRY5410) were significantly more heat shock-sensitive than their isogenic parent strain (JRY5413) (data not shown), the levels of Ras2 protein phosphorylation in these three strains were virtually indistinguishable (Fig. 3B). In addition, the steady-state level of Ras2p in these mutant strains was also unaffected (data not shown), demonstrating that neither decreased steady-state level of Ras2Val19p nor decreased Ras2Val19 protein phosphorylation was due to hyper-stimulation of the cAMP pathway per se.

Change in Intracellular Localization Did Not Effect Ras Protein Phosphorylation

Ras protein is localized at the plasma membrane and must be membrane-localized to promote growth. Mutations in RAS that prevent proper localization prevent stimulation of proliferation (33) although sufficiently high overexpression of localization-defective Ras protein can provide enough Ras function for viability. Therefore we tested whether the localization of Ras protein to the plasma membrane was necessary for its efficient phosphorylation.

Previous localization studies of Ras protein in yeast were of limited resolution due, at least in part, to the weak localization signal with existing antibodies in immunolocalization experiments. To better assay Ras protein localization, a fusion gene was constructed that joined the structural gene for green fluorescence protein (GFP) to the amino terminus of the coding region of RAS2. The GFP-RAS2 fusion gene was expressed from the strong constitutive GPD promoter and terminated with the PGK1 terminator (pJR1862). A fusion gene was also constructed in which the carboxyl-terminal cysteine codons of RAS2 (CCIIS-OH) were mutated to serine codons (SSIIS-OH) (pJR1863). The protein encoded by this mutated form of RAS2, ras2-SSIIS, cannot be processed at its carboxyl terminus and should not be membrane-localized.

Both GFP-Ras2 fusion proteins, GFP-Ras2 and GFP-Ras2-SSIIS, complemented the growth defect of the RAS null strain (data not shown). The strains expressing each of the GFP-Ras2 fusion proteins as their only functional Ras protein, ras1Delta ras2Delta pGFP-RAS2 (JRY5414) and ras1Delta ras2Delta pGFP-ras2-SSIIS (JRY5415), were visualized by fluorescence microscopy to determine the localization of the GFP-Ras2 fusion proteins. As expected, the GFP-Ras2 protein was primarily localized to the plasma membrane, whereas the GFP-ras2-SSIIS protein was localized throughout the cytoplasm (data not shown). There was no difference in Ras protein expression levels in these two strains as judged by immunoblot analysis (Fig. 4).


Fig. 4. Effect of Ras2 localization on Ras2p phosphorylation. Strains deleted for RAS1 and RAS2 were transformed with plasmids encoding GFP-Ras2 and GFP-Ras2-SSIIS fusion proteins. Left, strains expressing the fusion proteins were radiolabeled with 32P, and the Ras2 proteins were immunoprecipitated and analyzed for phosphorylation. Right, the strains were grown under the same conditions in the absence of radiolabel, and the Ras2 proteins were analyzed for steady-state protein level by immunoblot.
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To test whether phosphorylation of Ras2 protein was affected by its localization, the two strains were radiolabeled with 32P, and the GFP-Ras2 proteins in these cells were immunoprecipitated and analyzed for the extent of phosphorylation. There was no detectable difference in the levels of phosphorylation between the Ras protein that was localized and that which was not (Fig. 4). Thus membrane localization was not required for efficient phosphorylation of Ras protein in yeast.

Deletion of CDC25 Did Not Affect the Phosphorylation State of Ras Protein

Ras2Val19 protein, which is primarily GTP-bound, has a weaker interaction with Cdc25 guanine nucleotide exchange protein than does wild-type Ras2 protein, which is primarily GDP-bound (34). Because the GDP-bound form of Ras2 protein associates more tightly with Cdc25p, and because the GDP-bound form of Ras2p is more phosphorylated than predominantly GTP-bound Ras2Val19 protein, the Cdc25 dependence of Ras2 protein phosphorylation was evaluated.

The effect of a CDC25 deletion on the phosphorylation state of Ras protein was tested as above using the isogenic pair of strains RAS1 RAS2 cdc25Delta pCDC25-TRP1 pTPK1 (JRY5416) and RAS1 RAS2 cdc25Delta pTPK1 (JRY5417). There was no detectable difference in the level of phosphorylation of Ras proteins in cells with or without functional Cdc25 protein (Fig. 5). Therefore, association with the guanine nucleotide exchange factor was not necessary for the efficient phosphorylation or de-phosphorylation of Ras protein.


Fig. 5. Effect of CDC25 mutations on Ras2p phosphorylation. Left, a strain deleted for CDC25 and its parent were radiolabeled with 32P, and the Ras proteins in these cells were immunoprecipitated and analyzed by SDS-PAGE. Right, the Ras proteins from these cells were also analyzed for their steady-state level by immunoblot.
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Identification of a Phosphorylation Site on Ras2 Protein

The experiments described above established that Ras2p phosphorylation was dynamic and ruled out several possible explanations for why and how Ras2p phosphorylation was modulated. We then turned to a different approach for elucidating the role of Ras phosphorylation by evaluating phenotypes associated with mutant forms of Ras2p that lacked the ability to be efficiently phosphorylated. To construct such an allele, it was necessary to identify the phosphorylated residue(s) in Ras2p. As mentioned previously, Ras2 protein is phosphorylated only on one or more serine residues (19). However, there are 31 serine residues in mature Ras2p, and previous studies did not determine the number or stoichiometry of phosphorylation (19). The site(s) of Ras2p phosphorylation were determined through a combination of biochemical and genetic experiments and then the serines at these potential phosphorylation site(s) were changed to alanines. These studies were followed by in vivo analysis of phosphorylation-defective Ras2 proteins.

The predicted mass for Ras2 protein carrying 12 amino acids from the GST fusion, and accounting for post-translational farnesylation, proteolysis, and methyl esterification is 35,797 daltons (Da). The actual mass of the purified Ras2 protein was determined using electrospray mass spectrometry. Two protein species were detected in this sample. One protein, which represented 60-70% of the total protein, had a mass of 35,891.5 ± 2 Da. The other protein, which represented 30-40% of the total protein, had a mass of 35,971.4 ± 3 Da. These two masses differed by 80 Da, which is the mass of a phosphate. Thus, 60-70% of the Ras2 protein was phosphorylated. Surprisingly, the mass of the lower molecular weight species in this sample was 95 Da larger than the predicted mass of Ras2p. These 95 Da could have represented an additional phosphate (80 Da) plus an additional methylation (15 Da), or some other modification or combination of modifications that totaled 95 Da. However, as described below, it is unlikely that these 95 Da result in part from a second phosphorylation.

Identification of a Phosphorylated Ras2 Peptide

The endoproteolytic enzymes LysC, AspN, and ArgC were used to digest purified Ras2 protein, and the resulting peptides from these digestions were purified and subjected to analysis by mass spectrometry to identify phosphorylated peptides. Any phosphorylated peptide was expected to have a mass 80 Da larger than the mass predicted from the amino acid sequence of that peptide. Utilization of three different endoproteases allowed identification of numerous overlapping non-phosphorylated peptides which accounted for nearly the entire amino-terminal half of the Ras2 protein (Fig. 6) and suggested that the conserved amino-terminal half of the Ras2 protein was not phosphorylated in vivo.


Fig. 6. Phosphopeptide map of Ras2 protein. Masses for 20 different Ras2 peptides produced by proteolytic digestion with the endoproteases LysC, ArgC, and AspN were obtained by mass spectrometry (top panel) and used to construct a partial peptide map of Ras2 protein. A single phosphopeptide (Phosphopeptide 19) representing residues 194-254 was identified which contained 8 serines, 198, 202, 207, 214, 224, 225, 235, and 238. An additional non-phosphorylated peptide (Overlapping peptide 20) overlapped with the phosphopeptide-(194-254), thereby eliminating serines 224, 225, 235, and 238 as potential phosphorylation sites. Serine 6, 262, 273, 285, and 291 remained unaccounted for by mass data.
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Few peptides were isolated from the carboxyl-terminal domain of the Ras2 protein. However, one peptide fraction was a mixture of two peptides whose masses differed by 80 Da (Fig. 6, peptide 19). Approximately 60% of the protein in this peptide fraction had a mass of 6117 Da; the remaining 40% had a mass of 6197 Da. This ratio was similar to the ratio of the larger and smaller masses observed for the full-length Ras2 protein.

The mass of this phosphopeptide indicated that it represented amino acids 194-259 of Ras2p. This prediction was confirmed by amino-terminal sequence analysis of the peptide. There are 8 serines within this phosphopeptide. However, the measured mass of a peptide corresponding to residues 220-239 (Fig. 6, peptide 20), excluded the 4 serine residues in this peptide as sites of phosphorylation. The remaining 4 serine residues in the phosphopeptide (194-259), Ser-198, Ser-202, Ser-207, and Ser-214, were potential phosphorylation sites.

Mutagenesis of the Serines in Phosphopeptide 194-259

To determine whether the phosphorylation on peptide 194-259 was, in fact, the only phosphorylation on Ras2p, each of the 4 serine codons in this portion of the RAS2 gene was individually mutagenized to alanine codons. The resulting alleles, ras2Ala198 (pJR1864), ras2Ala202 (pJR1865), ras2Ala207 (pJR1866), and ras2Ala214 (pJR1867) were transformed on high copy TRP1-based plasmids into a RAS null strain (JRY5404), and all four mutants provided Ras function (data not shown). The resulting strains ras1Delta ras2Delta pras2Ala198 (JRY5418), ras1Delta ras2Delta pras2Ala202 (JRY5419), ras1Delta ras2Delta pras2Ala207 (JRY5420), and ras1Delta ras2Delta pras2Ala214 (JRY5421) were evaluated for Ras2 protein phosphorylation as described above and compared with the strain expressing wild-type RAS2 in high copy (JRY5430). Only the ras2Ala214 allele produced Ras2 protein that was less phosphorylated than wild-type protein (Fig. 7, compare lanes 6 and 7) suggesting that serine 214 was a phosphorylation site in Ras2 protein. The remaining three mutant proteins were phosphorylated to a level indistinguishable from that on wild-type protein (Fig. 7, compare lanes 3, 4, and 5 to lane 2). Steady-state level of Ras2 protein in all these strains was equivalent (Fig. 7, immunoblot).


Fig. 7. Analysis of Ras2 phosphopeptide serine to alanine mutants. Strains deleted for RAS1 and RAS2 and carrying a high copy plasmid encoding Ras2 protein with a serine to alanine mutation at positions 198, 202, 207, and 214 were radiolabeled with 32P, and the Ras2 proteins from these cells were immunoprecipitated and analyzed by SDS-PAGE (top panel). The strains were grown under the same conditions without radiolabel and analyzed by immunoblot to determine steady-state protein levels (bottom panels). Phosphorylation of Ras2Ala214 (**) protein was 56% of that on wild-type Ras2 protein.
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The extent of phosphorylation on Ras2Ala214p was 56% that of the wild-type protein. Therefore, substitution of serine 214 with a non-phosphorylatable alanine residue decreased the level of phosphorylation on full-length Ras2 protein by 44%. These data suggested that serine 214 was indeed a phosphorylation site for Ras2 protein. However, the residual phosphorylation of this mutant protein indicated that a second site was phosphorylated.

Search for the Second Phosphorylation Site in Ras2 Protein

Only 5 serine residues remained unaccounted for by peptide mass spectrometry data and were therefore still candidates for a second phosphorylation site: serine 6, serine 262, serine 273, serine 285, and serine 291 (Fig. 6). To determine whether one of these remaining serines was a second phosphorylation site, a series of site-directed mutants were constructed that eliminated various combinations of these and other serine residues (Fig. 8A). All of the mutant alleles were transformed into a RAS null strain (JRY5404) on high copy TRP1-marked plasmids, and all were able to complement the growth defect of a null strain (data not shown).


Fig. 8. Analysis of Ras2 multiple serine to alanine mutants. A (top panel), strains deleted for RAS1 and RAS2 and carrying a high copy plasmid encoding Ras2 protein with multiple serine to alanine mutations were radiolabeled with 32P, and the Ras2 proteins from these cells were immunoprecipitated and analyzed by PAGE for phosphorylation. A (lower panel), the strains were grown under the same conditions without radiolabel and analyzed by immunoblot to determine the steady-state protein levels. B, (top panel), the Ras null strain was also transformed with plasmids with sequentially more serine to alanine mutations, radiolabeled with 32P, and the Ras proteins immunoprecipitated and analyzed by SDS-PAGE for phosphorylation. B (lower panel), the strains were grown under the same conditions without radiolabel to determine steady-state protein by immunoblot.
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Evaluation of protein phosphorylation of these mutant Ras2 proteins revealed several points of interest (Fig. 8A). Mutation of serine residue 6, 262, 273, 285, or 291 to alanine had no effect on the level of phosphorylation of these Ras2 mutant proteins (Fig. 8A, lanes 7, 9, 12, and 14). Therefore, none of these serine residues was likely to serve as a phosphorylation site in vivo. In all cases, a decrease in the level of Ras2 phosphorylation occurred only in combinations containing the S214A mutation (Fig. 8A, lanes 8 and 13). In addition, all Ras2 proteins with the S214A mutation were less phosphorylated (Fig. 8A, lanes 8 and 13), demonstrating that serine 214 was a preferred site of phosphorylation in vivo.

Interestingly, when additional serine or threonine residues near serine 214 were mutated to alanine in cis to the S214A mutation, the level of Ras2 protein phosphorylation could be reduced below the level observed when the S214A mutation was present alone (Fig. 8B). The greater the number of serine (or threonine) to alanine changes in cis to S214A, the lower the level of phosphorylation on Ras2p (Fig. 8B, compare lanes 3, 4 and 5). Thus, although serine 214 was the preferred phosphorylation site for Ras2p, mutation of serine 214 to alanine resulted in the phosphorylation of other serine or threonine residues near serine 214.

Characterization of Cells Expressing the ras2Ala214 Allele

The identification of a preferred phosphorylation site for Ras2p and subsequent mutagenesis of this serine residue to alanine allowed testing of what phenotype(s) was associated with reduction of Ras2p phosphorylation levels. TRP1-marked versions of five different alleles of RAS2 (ras2Ala214, RAS2Val19-Ala214, ras2Ala198-Ala202-Ala207-Ala214-Ala235-Ala238, RAS2, RAS2Val19) were integrated at the TRP1 locus in a ras1Delta ras2Delta null strain (JRY5404) to produce an otherwise isogenic set of strains (JRY5422, JRY5423, JRY5424, JRY5425, and JRY5426, respectively). Analysis of growth rate of these five integrated strains demonstrated that presence of a phosphorylation mutation in RAS2 or in cis to the activating mutation RAS2Val19 had no effect on the doubling time of these strains (Table II). Thus neither phosphorylation of Ras2p nor the serine residues at these positions contributed substantially to the essential function of Ras2p under these conditions. However, this assay was relatively insensitive to decreases in Ras2 function, because strains with less than 5% wild-type Ras2p levels grow at normal rates.2

Table I. Strains


Strain Genotype

JRY5404 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1857 (TPK1-  URA3-2µ)
JRY5430 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1244 (RAS2-TRP1-2µ)
JRY5405 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1859 (RAS1-  TRP1-2µ)
JRY5406 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1860 (RAS1val19-  TRP1-2µ)
JRY5470 MATalpha ade2-1 his3 leu2 trp1 ura3 ras1::HIS3 ras2Delta ::LEU2 + pJR1912 (RAS2val19-TRP1-2µ)
JRY5409 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1244 (RAS2-  TRP1-2µ) + pJR1857 (TPK1-URA3-2µ)
JRY5407 MATalpha ade2 ade3 his3 leu2-3,112 trp1 ura3 ras1Delta
JRY5408 MATalpha ade2 ade3 his3 leu2-3,112 trp1 ura3 ras1Delta  + pJR1861 (RAS2val19-URA3-ADE3-CEN)
JRY5410 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 bcy1Delta ::LEU2 + pJR1244 (RAS2-  TRP1-2µ)
JRY5414 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1862 (GFP-RAS2-  TRP1-2µ)
JRY5415 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1863 (GFP-RAS2-  SSIIS-TRP1-2µ)
JRY5416 MATalpha leu2 trp1 ura3 cdc25Delta ::LEU2 + pCDC25-TRP1 + pJR1857 (TPK1-URA3-2µ)
JRY5417 MATalpha leu2 trp1 ura3 cdc25Delta ::LEU2 + pJR1857 (TPK1-URA3-2µ)
JRY5429 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pGST-RAS2-TRP1-2µ)
JRY5418 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1864 (ras2Ala198-TRP1-2µ)
JRY5419 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1865 (ras2Ala202-TRP1-2µ)
JRY5420 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1866 (ras2Ala207-TRP1-2µ)
JRY5421 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1867 (ras2Ala214-TRP1-2µ)
JRY5431 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1883   (ras2ala198,202,207,308,311,313,315-TRP1-2µ)
JRY5432 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1884 (ras2Ala198,202,207,224,225-  TRP1-2µ)
JRY5433 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1885 (ras2Ala202,207,235,238-  TRP1-2µ)
JRY5434 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1886 (ras2Ala202,207,224,225-  TRP1-2µ)
JRY5435 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1887 (ras2Ala198,202,207-  TRP1-2µ)
JRY5436 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1888 (ras2Ala6,198,202,207-  TRP1-2µ)
JRY5437 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1889 (ras2Ala202,207,214,224,225-  TRP1-2µ)
JRY5438 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1890 (ras2Ala285,291   -TRP1-2µ)
JRY5439 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1891 (ras2Ala262,263TRP1-2µ)
JRY5440 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1892   (ras2Ala198,202,207,214,262,263-TRP1-2µ)
JRY5441 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1893 (ras2Ala273-TRP1-2µ)
JRY5442 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1894   (ras2Ala202,207,224,225,235,238-TRP1-2µ)
JRY5443 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1895   (ras2Ala198,202,207,214,308,311,313,315-TRP1-2µ)
JRY5444 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1896   (ras2Ala198,202,207,214,235,238-TRP1-2µ)
JRY5445 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1897   (ras2Ala202,207,214,224,225,226,227,235,238-TRP1-2µ)
JRY5422 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1872-EcoRV   (ras2Ala214::TRP1)
JRY5423 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1873-EcoRV   (ras2Val19ala214::TRP1)
JRY5424 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1874-EcoRV   (ras2Ala198,202,207,214,235,238::TRP1)
JRY5425 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1875-EcoRV (RAS2::TRP1)
JRY5426 MATalpha ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 ras1Delta ::HIS3 ras2Delta ::LEU2 + pJR1876-EcoRV   (RAS2Val19::TRP1)

Table II. Characterization of phosphorylation site mutants


Strain Doubling time YPD % survival 90-s heat shock

h
JRY5425 RAS2 integrated 1.5 73
JRY5422 ras2ala214 integrated 1.5 56
JRY5424 ras2ala198,202,207,214,235,238 integrated 1.5 64
JRY5426 RAS2val19 integrated 1.5 ] 0.1 
20 × 
JRY5423 RAS2val19ala214 integrated 1.5 2

A widely used measure of RAS2Val19 function is the ability of this mutation to render strains highly sensitive to heat shock. Interestingly, combining the serine 214 to alanine mutation with the activating RAS2Val19 mutation resulted in a 20-fold increase in heat shock resistance (Table II). This result suggested that a decrease in Ras2 protein phosphorylation led to a detectable decrease in signaling through the Ras-cAMP pathway.

Growth on Other Media

The five integrated strains were tested for growth on multiple different media and at different temperatures. This analysis included media containing drugs known to affect cAMP production in other organisms (caffeine) and that block synthesis of isoprenoids (lovastatin), which are necessary for the post-translational processing of Ras proteins. However, these components had no differential effect on growth of strains expressing the five different alleles (data not shown). However, a differential effect on growth was observed among these five strains when they were grown on YPD medium containing formamide, which is an increasingly popular reagent for identifying conditional alleles in yeast (35). Cells containing Ras2Val19 protein grew more poorly on medium containing formamide than cells with wild-type Ras2 protein (Fig. 9). Strains expressing phosphorylation-defective alleles of Ras2 protein grew better on medium containing formamide than the wild-type strain (Fig. 9). In addition, an isogenic strain expressing only the ras2-23 temperature-sensitive allele of RAS2 also grew better on formamide than the wild-type strain. Together, these data suggested that formamide sensitivity provided a sensitive phenotypic read-out of Ras signaling as follows: increases in signaling through the Ras-cAMP pathway (expression of Ras2Val19) led to an increased sensitivity to formamide, and decreases in signaling through the Ras-cAMP pathway (expression of ras2-23 or a phosphorylation-defective mutant Ras protein) led to decreased formamide sensitivity. These results were consistent with the suppression of heat shock sensitivity observed when the S214A mutation was present in cis to the RAS2Val19 mutation.


Fig. 9. Analysis of formamide sensitivity of wild-type and phosphorylation mutant RAS2 alleles. Strains deleted for RAS1 and RAS2 were transformed with wild-type, activating, and various phosphorylation mutant alleles of RAS2 on integrating plasmids, and the resulting cells were grown to mid-log phase. Cells were plated in 10-fold serial dilutions onto YPD containing 3% formamide and YPD alone and incubated at 30 °C to compare growth.
[View Larger Version of this Image (74K GIF file)]

Because phosphorylation has been shown to alter the localization of some proteins, we tested whether mutations in the phosphorylation site(s) affected the localization of Ras2p. Proteins from the five strains above were fractionated into membrane and cytosolic pools. The localization of the Ras2 proteins in these cells was then analyzed by immunoblot. Reduction of phosphorylation had no effect on the localization of Ras2 protein (data not shown).


DISCUSSION

This study provided a systematic evaluation of phosphorylation of Ras1p and Ras2p of S. cerevisiae with a focus on Ras2p. Both Ras1p and Ras2p were found to be phosphorylated, and at least in the case of Ras2p, the phosphorylation was dynamic and occurred on roughly 60% of the Ras proteins. Studies of the half-life of Ras2p and of the half-life of its phosphorylation established that Ras2p phosphorylation was dynamic and potentially served a regulatory role. Interestingly, activated forms of both Ras1p and Ras2p were hypophosphorylated, and these proteins were present at only 10-20% the level of wild-type Ras protein.

We explored potential links between Ras protein phosphorylation and the role of Ras protein in nutrient sensing and signaling through the Ras-cAMP pathway. Multiple lines of evidence suggest the activity of the Ras-cAMP pathway is under tight feedback control. Intracellular levels of cAMP are unusually high when the activities of the cAMP-dependent protein kinases are lowered by mutation. In addition a lower level of cAMP is found in cells with an elevated level of a cAMP-dependent protein kinase (22). Both of these effects are dependent on the presence of RAS and CDC25. Because the modulation of cAMP levels in response to mutations of the cAMP-dependent protein kinase is dependent on RAS, we tested whether modulation of cAMP levels involved phosphorylation of Ras2p in a feedback loop by the cAMP-dependent protein kinases. However, in this study, several lines of evidence indicated that the difference between the phosphorylation of wild-type Ras2p and the hypophosphorylated Ras2Val19 protein did not reflect the activity level of the cAMP response pathway.

Because Cdc25p is partially responsible for regulating the ratio of GTP- and GDP-bound Ras proteins in yeast cells, and because GTP-bound Ras protein associates more poorly with Cdc25p than does GDP-bound Ras protein, we tested whether phosphorylation of Ras2 protein was modulated through an interaction with Cdc25p. The level of phosphorylation of Ras protein was not affected by the presence or absence of CDC25. Thus, Ras2p did not need association with Cdc25 protein to become phosphorylated.

The proper localization of Ras2 protein to the plasma membrane was not necessary for its phosphorylation, as localization-defective alleles of Ras2 protein were phosphorylated to a level indistinguishable from wild-type. However, because GFP-Ras2-SSIIS protein was sufficient to promote growth when present at elevated levels, presumably some of the mutant protein was, in fact, reaching the plasma membrane. Although it was possible that some of the Ras2-SSIIS protein was phosphorylated during transient associations with the membrane, if this were the mechanism by which the Ras2-SSIIS protein became phosphorylated, it is unlikely that the level of phosphorylation on the mutant and wild-type proteins would have been the same.

We anticipated that an allele of RAS2 that encoded a protein unable to be phosphorylated would provide insight into any regulatory role of the phosphorylation. The site of Ras2p phosphorylation was determined by a combination of peptide mapping by mass spectrometry and site-directed mutagenesis. These studies indicated only a single site of Ras2p phosphorylation and that approximately 60% of the Ras molecules were phosphorylated. The serine at position 214 was the preferred site of phosphorylation; however, the protein kinase(s) responsible for Ras2p phosphorylation was able to phosphorylate a variety of serine residues in the vicinity of position 214 when this site was altered. This region of the Ras2 protein of yeast is missing from mammalian Ras, for which three-dimensional structural information is available, precluding insight into the structural basis of this phosphorylation flexibility.

The phosphorylation of Ras2p was unaffected by changes in the activity of the Ras-cAMP response pathway as described above. However, changes in the phosphorylation state of Ras2p appeared to affect signaling through the Ras-cAMP pathway: phosphorylated forms of Ras2p appeared to be more active than non-phosphorylated forms based upon the phenotypes of mutant proteins that were significantly reduced for phosphorylation. Presumably Ras2Val19 protein is hyperactive despite its decreased phosphorylation state, because it is primarily bound to GTP and therefore relieved from modulation by phosphorylation. We found two phenotypes associated with decreases in Ras2 protein phosphorylation. The first was the 20-fold increase in survival upon heat shock of cells expressing the RAS2Val19-Ala214 allele compared with cells expressing RAS2Val19. Thus a single point mutation that led to a decrease in phosphorylation led to a detectable decrease in signaling through the Ras-cAMP pathway. The second phenotype was a decrease in sensitivity to formamide of strains harboring mutant Ras2 protein with significantly reduced phosphorylation. Formamide is thought to destabilize protein-protein interactions by disrupting hydrogen bonding (35). It is therefore possible that the phosphorylation on Ras2 protein is affecting the interaction of Ras2p with another protein(s) in the Ras signaling cascade, perhaps with the cyclase-associated protein CAP (see below). Because activated alleles of RAS were more sensitive to formamide, and because cells expressing a hypomorphic allele of RAS, ras2-23, were more resistant to formamide, formamide sensitivity in this context was a phenotypic readout for Ras signaling. If so, a decrease in phosphorylation led to a decrease in signaling through the Ras-cAMP pathway. Although we cannot rule out the possibility that any protein with six point mutations might exhibit a similar phenotype, the observation that a single point mutation at position 214, which reduces the phosphorylation of Ras2p by only 40%, can suppress the heat shock sensitivity of RAS2Val19 by 20-fold, suggested that it was the decrease in phosphorylation and not merely six substitutions that led to an altered phenotype.

The subtlety of these phenotypes suggested that phosphorylation of Ras2p may modulate Ras function but was not necessary for growth. Moreover, the phosphorylation of Ras2p occurred on a residue in the hypervariable domain of Ras2p which is dispensable for viability. However, we could not exclude the possibility that phosphorylation of Ras protein was essential, but only a small quantity of phosphorylated protein was sufficient for viability, because even in strains with multiple serine to alanine mutations there remained a detectable pool of phosphorylated protein.

A full cellular response to Ras2Val19 protein is dependent upon the adenylyl cyclase-associated protein (CAP), whereas CAP is not necessary for wild-type Ras function (see below) (36). CAP is a multifunctional protein; the amino-terminal third binds to adenylyl cyclase and has been implicated in activation of the cAMP-dependent protein kinase pathway. The carboxyl-terminal domain of CAP binds monomeric actin and serves a cytoskeletal regulatory function. In addition, CAP contains a short proline-rich domain between the Ras-related and cytoskeletal-related portions of the protein, which interacts with other proteins including yeast actin-associated protein Abp1p, and with cortical actin-containing structures (37). These observations suggest CAP may coordinate Ras-cAMP signaling and cytoskeletal organization.

Deletion of SRV2, which encodes CAP, is lethal in some strain backgrounds. Alleles of SRV2 that are deleted for the amino-terminal Ras-related portion of the protein are viable but suppress some phenotypes associated with activated RAS. In fact, alleles of SRV2 that encode proteins deleted for the RAS-responsive portion of CAP significantly decrease the ability of Ras2Val19 protein to stimulate the production of cAMP (38). However, elimination of the same portion of SRV2 has no effect on the ability of wild-type Ras2 protein to stimulate the production of cAMP, suggesting that activated and non-activated alleles of Ras protein interact differently with CAP (36). Perhaps the phosphorylation of Ras protein is related to its interaction with CAP. RAS also plays a role in the completion of mitosis in addition to its essential role in the G1 phase of the cell cycle (39). It is possible that the phosphorylation of Ras protein plays a role in the mitotic function of Ras protein. There has been no analysis of altered mitotic function(s) of activated alleles of RAS.

Finally, we address the mass discrepancy identified in this study. Our measurements of the mass of Ras2p exceeded the calculated molecular mass of the protein by 175 daltons. As described above, 80 daltons were accounted for by the phosphorylation of Ras2p on serine 214. At present, we do not know the basis of the remaining 95-dalton discrepancy. One possibility is that the reported sequence of Ras2 contains errors that add up to 95 daltons. However, we have sequenced the cloned RAS2 used in these studies and confirmed the published sequence. A second phosphorylation accounting for 80 of the remaining 95 daltons is inconsistent with our data. At present we favor the view that the 95 daltons represent one or more previously uncharacterized modifications of Ras2p which, based upon the confirmed mass of several overlapping Ras2 peptides from the conserved amino-terminal domain, presumably occurs in the hypervariable domain of the protein.


FOOTNOTES

*   This work was supported by Grant IRT-26 from the Tobacco-related Disease Research Program and by National Institutes of Health Grant GM35827 (to J. R.). Additional core support was provided by an NIEHS Mutagenesis Center Grant P30 ESo 1896.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.
Dagger    Supported by a Merck predoctoral fellowship and NIH Training Grants GM07232 and NIH ES07075. Current address: Nina Ireland Laboratory of Developmental Neurobiology, Dept. of Psychiatry and Langley Porter Psychiatric Inst., 401 Parnassus Ave., University of California, San Francisco, CA 94143.
§   To whom correspondence should be addressed: Div. of Genetics, Dept. of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720. Tel.: 510-642-7047; Fax: 510-642-6420; E-mail: jasper{at}mendel.berkeley.edu.
1   The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); oligo, oligonucleotide; GST, glutathione S-transferase; CAP, adenylyl cyclase-associated protein; PAGE, polyacrylamide gel electrophoresis; 5-FOA, 5-fluoroorotic acid; GFP, green fluorescence protein.
2   J. L. Whistler and J. Rine, unpublished observation

ACKNOWLEDGEMENTS

We thank David King for his tireless efforts at the mass spectrometer during the peptide analysis stage of these studies and without whom the phosphorylation site would not have been mapped. We also thank Sharlene Zhou and Michael Moore who provided peptide sequence. We thank Cindy Trueblood for her contributions to strain and plasmid constructions described in this work. We thank Randy Hampton and Stig Hansen for stimulating discussions and their excellent suggestions. We thank the members of the lab for their support.


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