(Received for publication, June 9, 1995; and in revised form, August 17, 1995)
From the
The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo. The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo. Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells. One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1. p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family. The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1. Site-directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3. Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine. Fpr3 represents the first phosphotyrosyl protein identified in S. cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1.
Phosphotyrosine-specific phosphoprotein phosphatases (PTPs) ()have been identified in many evolutionarily divergent
eukaryotes. These enzymes form a distinct superfamily and are unrelated
in sequence to serine/threonine-specific phosphoprotein phosphatases
(for reviews, see (1, 2, 3) ). All PTPs
possess stretches of sequence similarity within their catalytic
domains, including the active site consensus sequence
(I/V)HCXAGXGR(S/T)G. This hallmark sequence contains
an invariant Cys residue, which acts as the nucleophile during the
dephosphorylation reaction, and a GXGXXG motif, which
forms a phosphate-binding loop and is also found in nucleotide-binding
proteins such as protein kinases and GTPases(4) . The
substrate-binding cleft of PTPs is surrounded by basic amino acids,
which may explain the preference for acidic residues near the
phosphorylated tyrosines in PTP substrates(5, 6) .
In the budding yeast, Saccharomyces cerevisiae, dedicated
tyrosine-specific protein kinases have not been identified. However, a
number of genes encoding PTPs have been reported. These PTPs include
both phosphotyrosine-specific and dual-specific enzymes as seen in
higher eukaryotes. Two of the S. cerevisiae PTPs appear to be
MAP kinase phosphatases. The dual-specific PTP encoded by the MSG5 gene dephosphorylates Fus3 and thereby contributes to the reversal
of pheromone arrest(7) . The PTP2 gene product is
thought to dephosphorylate Hog1, a MAP kinase involved in
osmoregulation(8) . At least two S. cerevisiae PTPs
are involved in cell cycle control: the CDC14 gene product is
required for progression through S phase(9) , and the product
of the MIH1 gene, the S. cerevisiae homolog of the
fission yeast cdc25, is thought to
dephosphorylate the Cdc28 kinase(10) . The YVH1 PTP gene is
induced by nitrogen starvation and encodes a PTP that is required for
maximal growth(11) .
PTP1, the first PTP gene
reported in budding yeast, was identified by the polymerase chain
reaction using oligonucleotides corresponding to conserved PTP
catalytic domain sequences as primers (12) . Ptp1 appears to be
phosphotyrosine specific and is comprised of a carboxyl-terminal
catalytic domain and a unique 55-residue amino-terminal region of
unknown function. Although Ptp1 is active in vitro against
artificial substrates, the physiological role of Ptp1 is unknown; PTP1 disruption or overexpression does not overtly effect
growth at extreme temperatures, sensitivity to different metal ions,
osmotic stability, carbon source utilization, mating, or
sporulation(12, 13) . ()However, expression
of PTP1 in fission yeast mimics cdc25
overexpression and leads to precocious mitosis(14) . In
addition, overexpression of PTP1 in S. cerevisiae rescues the synthetic lethality resulting from disruption of both PTP2 and PTC1, a gene encoding a putative
Ser/Thr-specific phosphoprotein phosphatase of the PP2C
class(15) . These results suggest that when overproduced, Ptp1
may be capable of dephosphorylating Cdc2 and Hog1, but the relevance of
these activities to normal Ptp1 function is unclear.
Here, we describe the identification of yeast phosphotyrosyl proteins that are dephosphorylated by Ptp1 in vivo and present evidence that one Ptp1 substrate is the nucleolar immunophilin, Fpr3.
To induce transcription of genes driven
by a GAL promoter, cultures were grown overnight to A = 1 in defined medium containing 2%
raffinose. Galactose was then added to a final concentration of 2%, and
the cells were grown for an additional 3 h prior to harvesting. For
large scale purification of p70
, strain PJ58-2B (ptp1
ptp2
mih1
) was grown in YPD in a
200-liter fermenter with vigorous aeration to stationary phase (A
= 3.5).
Immunoblot
analyses were carried out as described (21) with the following
modifications. Cell proteins (100 µg of protein/lane) were
fractionated by SDS-PAGE and transferred to a polyvinylidene-difluoride
membrane (PVDF) (Immobilon P, Millipore). Blocking buffer containing 3%
bovine serum albumin, 0.1% Tween 20, 0.5 M NaCl, 0.5% Nonidet
P-40, and 50 mM Tris-HCl (pH 7.5) was used for all blocking,
antibody incubation, and rinsing steps. The following antibodies were
used at a concentration of 1 µg/ml to probe immunoblots:
anti-phosphotyrosine monoclonal antibody (mAb) 4G10 (22) (Upstate Biotechnology), mAb FB2(23) , mAb
6G9(24) , a polyclonal rabbit anti-phosphotyrosine antibody
prepared by the method of Kamps and Sefton(25) , rabbit
anti-Fpr3 serum(18) , anti-Src mAb 2-17 (Microbiological
Associates, Rockville, MD), and anti-Myc mAb 9E10 (26) .
Primary antibodies were detected by incubation with appropriate
horseradish peroxidase-conjugated anti-immunoglobulin antibodies
(Pierce), followed by chemiluminescence detection with
Renaissance enhanced luminol reagent (Dupont NEN) and
exposure of Kodak X-AR film. Immunoblot signals were quantified by
densitometry using a ScanMaker MRS-600ZS
(Microtek,
Taiwan).
The wild-type and mutant PTP1 genes were cloned into the glutathione S-transferase (GST) fusion vector PGEX-3X (Pharmacia Biotech Inc.). In preparation for these ligations, an adapter, 5`-CGGGATCCAAATGCAGGCCTCTCGAGATCGATGAATTC-G3`, which contains a BamHI site (boldface type) followed by the first 7 translated nucleotides of PTP1 (underlined), and StuI, ClaI, and EcoRI sites (italicized) was first inserted between the BamHI and EcoRI sites in the vector. This strategy allowed the in-frame insertion of a PvuII-ClaI fragment containing the remainder of PTP1 (nucleotides 8-3042 excised from YEp51-PTP1) into the modified vector between the StuI and ClaI sites. The resulting constructs encode GST-Ptp1 and GST-Ptp1(C252A) fusion proteins with a factor Xa cleavage site between the GST and Ptp1 coding segments. Plasmid YEp352GAL-v-src was described previously (28) .
FPR3 expression plasmids YEp351-FPR3myc (pYB1010), YEp351GAL-FPR3myc (pYB124), YEp351GAL-FPR3 (pYB123), YEp351GAL-FPR3N (pYB126), YEp351GAL-FPR3C (pYB120), and pGXFPR3A (encoding GST-Fpr3N) are described in (18) . Plasmids expressing mutant derivatives of FPR3 were generated by PCR using a pUC19-derived plasmid containing wild-type FPR3 (pNH2.2; described in (18) ) as template. A double mutant (Y184F,Y189F) was generated using two PCR primers, each of which contained both a change in codon 184 from TAT to TTT (nucleotides 550-552) and in codon 189 from TAC to TTC (nucleotides 265-267). Primer 1 spanned nucleotides 546-573 on the coding strand, and primer 2 spanned nucleotides 573-540 on the noncoding strand. In one PCR reaction, primer 1 was used with an additional downstream primer, spanning nucleotides 839-857 (noncoding strand), to generate a 311-bp product. In a separate reaction, primer 2 was used with an upstream primer, spanning nucleotides 363-382 (coding strand), to generate a 210-bp product. In the final PCR reaction, an overlap extension, the 2 initial overlapping products were purified and used as template primers together with the upstream and downstream primers. This reaction generated a 494-bp product spanning nucleotides 363-857 with mutations at codons 184 and 189. The product was digested with BspE1 and EcoRI to generate a 455-bp fragment. This fragment was ligated to the 4.5-kilobase fragment of BspE1 and EcoRI-digested pNH2.2, thus replacing the corresponding region of wild-type FPR3. To generate versions of FPR3 containing each of the single mutations, Y184F and Y189F, a similar procedure was used, except that the primers ``1'' and ``2'' were changed to include a mutation only at codon 184 for Y184F and only at codon 189 for Y189F. To express the mutated FPR3 genes in yeast, they were excised from pNH2.2 with AflIII and HindIII, and the AflIII site was filled in with the Klenow fragment of DNA polymerase I. The resulting fragments were then ligated into the vector YEp351GAL(18) , which had been opened with SalI, treated with Klenow, and then cut with HindIII. All constructs were verified by DNA sequencing.
S-Sepharose fractions enriched for
p70 (300 ml total) collected from the five 100-g batches of yeast were
brought to a final concentration of 1% Triton X-100, dialyzed twice for
2.5 h against 4 liters of buffer C containing 0.1% Triton X-100, and
precleared by incubation with 0.5 ml of protein A-Sepharose CL-4B
(Pharmacia) for 4 h at 4 °C; the protein A-Sepharose was removed by
centrifugation at 4000 g for 5 min. The resulting
supernatant solution was mixed with 1.0 ml of protein A-Sepharose, to
which had been coupled anti-phosphotyrosine mAb FB2(23) , and
incubated with gentle rocking at 4 °C for 6 h. The beads were
washed with 10 ml of buffer C containing a final concentration of 120
mM NaCl, and p70 was eluted with 2 ml of 60 mM
phenylphosphate. The eluted protein was precipitated with 10%
trichloroacetic acid, treated with 10 mM 4-vinylpyridine to
alkylate Cys residues, resolved by SDS-PAGE, and transferred to
Immobilon-P. Ponceau S staining revealed a single band in the 70-kDa
size range. This procedure yielded approximately 15 µg of p70 from
500 g of yeast. The band was excised and digested with sequencing grade
trypsin (Boehringer Mannheim). Tryptic peptides were separated by
reverse phase chromatography (Brownlee C8 column, 1
250 mm,
Applied Biosystems) using a 172A microbore high pressure liquid
chromatograph (Applied Biosystems) and subjected to microsequencing by
Edman degradation in a 477A protein sequencer (Applied Biosystems,
Inc.).
Figure 1:
Ptp1 has broad substrate
specificity in vivo and in vitro. A wild-type strain,
PJ55-16A (lanes 1, 3, 5), and the ptp1 strain PJ55-16C (lanes 2, 4, 6) were transformed with YEp352GAL-v-src.
Transformants were grown in the presence of galactose to induce
expression of v-src. The cells were lysed, and lysates were
incubated with buffer (lanes 1, 2) or GST-Ptp1 (lanes 3, 4) or with catalytically inactive
GST-Ptp1(C252A) (lanes 5, 6) and analyzed by
immunoblotting with anti-phosphotyrosine mAb 4G10 (upper
panel) or with anti-p60
mAb 2-17 (lower panel, lanes 1, 2). Molecular mass
(in kDa) markers are indicated on the left.
To determine whether Ptp1 can dephosphorylate in
vitro the proteins phosphorylated by
p60, a bacterially expressed GST-Ptp1 fusion
protein was incubated with the phosphotyrosyl proteins in lysates from
either PTP1 or ptp1
cells expressing
v-src. The level of tyrosine phosphorylation was drastically
reduced by incubation with GST-Ptp1 (Fig. 1, lanes 3 and 4). In contrast, incubation with catalytically
inactive GST-Ptp1(C252A) did not significantly reduce the level of
tyrosine phosphorylation (Fig. 1, lanes 5 and 6). This result confirms that Ptp1 has broad substrate
specificity in vitro.
Figure 2:
PTP1 disruption results in enhanced
tyrosine phosphorylation of several proteins. Lysates of strain
PJ58-2B (ptp1 ptp2
mih1
) (lanes
1, 3, 5, 7) and its congenic wild-type
strain PJ58-8B (lanes 2, 4, 6, 8) were analyzed by immunoblotting with anti-phosphotyrosine
mAbs 4G10 (lanes 1, 2), FB2 (lanes 3, 4), and 6G9 (lanes 5, 6) and with
anti-phosphotyrosine polyclonal antibodies (R
PY, lanes 7, 8). Arrowhead indicates migration of
p70.
To determine whether the activity of other S. cerevisiae PTPs affected the tyrosine phosphorylation state of p70 (or any other protein) during normal growth, lysates of strains containing disruptions in PTP1, PTP2, and MIH1 were compared by anti-phosphotyrosine antibody immunoblotting. Anti-phosphotyrosine antibody recognized p70 only in strains disrupted for PTP1 (Fig. 3A). Disruption of PTP2 or MIH1 did not result in a detectable increase in the level of tyrosine phosphorylation on p70 or in the appearance of additional phosphotyrosyl proteins (Fig. 3A, lanes 3 and 4). These results suggest that phosphotyrosyl p70 is dephosphorylated only by Ptp1. In addition, these results support the conclusion that Ptp2, which is thought to dephosphorylate the Hog1 kinase(8) , and Mih1, which is thought to dephosphorylate the Cdc28 kinase(10) , have more restricted substrate specificities than Ptp1.
Figure 3:
PTP1
expression suppresses tyrosine phosphorylation of p70. Panel
A, effect of disruptions in PTP1, PTP2, and MIH1 on protein tyrosine phosphorylation. The wild-type strain
PJ55-16A (lane 1), the ptp1 strain
PJ55-16C (lane 2), the ptp2
strain
PJ55-16D (lane 3), and the ptp1
ptp2
mih1
strain PJ58-2B (lane 4) were lysed by
exposure to NaOH as described under ``Materials and
Methods.'' Lysates were analyzed by immunoblotting with
anti-phosphotyrosine mAb 4G10. Panel B, restoration of PTP1 expression in ptp1
yeast suppresses p70
tyrosine phosphorylation. The ptp1
strain, PJ55-16C (lane 1), strain PJ55-16C expressing YEp51-PTP1 (lane 2), and wild-type strain PJ55-16A (lane
3) were grown in the presence of galactose and lysed by agitation
with glass beads. Lysates were analyzed by immunoblotting with
anti-phosphotyrosine mAb 4G10.
To
determine whether Ptp1 could dephosphorylate p70 in vitro,
lysates from the ptp1 ptp2
mih1
triple mutant
strain were incubated with GST-Ptp1 or GST-Ptp1(C252A). Incubation with
GST-Ptp1 resulted in the complete dephosphorylation of p70 (Fig. 4, lane 3), while GST-Ptp1(C252A) had no effect (Fig. 4, lane 4). The same results were obtained with
soluble Ptp1 preparations generated by cleavage from the GST carrier by
digestion with factor Xa (data not shown). This experiment suggests
that p70 is a direct substrate of Ptp1 but does not exclude the
possibility that Ptp1 activates another protein-tyrosine phosphatase,
which in turn dephosphorylates p70. The results of subsequent
experiments (see below) provide evidence that p70 is a direct substrate
of Ptp1.
Figure 4:
Dephosphorylation of p70 by Ptp1 in
vitro. Lysates of the wild type strain PJ58-8B (lane
1), the ptp1 ptp2
mih1
strain
PJ58-2B (lanes 2-4), or p70-enriched S-Sepharose
fractions from strain PJ58-2B (lanes 5-7) were
incubated with buffer (lanes 1, 2, 5),
GST-Ptp1 (lanes 3, 6), or GST-Ptp1(C252) (lanes
4, 7) and then boiled in SDS sample buffer and analyzed
by immunoblotting with anti-phosphotyrosine mAb 4G10. The 65-kDa
GST-Ptp1 fusion proteins (lanes 3-7) are stained
nonspecifically by the 4G10 antibody.
Figure 5:
Purification of p70. Panel A,
anti-phosphotyrosine immunoblot analysis of initial cell lysate (lane 1) and p70 peak fractions from the following
purification steps: 1 M NaCl eluate from 100,000 g pellet (lane 2), NaCl eluate from S-Sepharose column (lane 3), phenyl-phosphate eluate from anti-phosphotyrosine
mAb FB2-coupled Sepharose (lane 4). Panel B,
two-dimensional electrophoresis of eluate from anti-phosphotyrosine
resin (100 ng of protein). Proteins in the gel were transferred to PVDF
membrane and gold stained (left) and then stained with
anti-phosphotyrosine antibodies (right) as described under
``Materials and Methods.''
Tryptic peptides were generated
from purified p70, and five were sequenced. Four of the five sequences
matched precisely the amino acid sequence of S. cerevisiae Fpr3, a recently identified nucleolar FK506-binding protein (Fig. 6)(18, 34, 35) . FK506-binding
proteins (FKBPs) are immunophilins that bind the structurally related
immunosuppressive drugs, FK506 and rapamycin. The formation of
complexes between these drugs and the predominant cytosolic FKBP,
FKBP-12, inhibits signal transduction pathways in both vertebrates and
yeast, but the normal functions of these proteins are unknown (see
``Discussion''). Genetic analysis confirmed that p70 is
identical to Fpr3. When a ptp1 strain was transformed
with a high copy plasmid expressing galactose-inducible FPR3,
p70 was greatly overproduced; conversely, p70 was completely absent in
the ptp1
fpr3
strain (Fig. 7A).
These findings, together with the sequence of the tryptic peptides
derived from p70, verify that p70 is Fpr3. The anomalous
electrophoretic mobility of Fpr3 (calculated molecular mass, 47 kDa)
has been noted previously(18) .
Figure 6: Purified phosphotyrosyl p70 is a phosphorylated form of Fpr3. The deduced amino acid sequence of the FPR3 gene product is shown in the one-letter code(18) . The sequence of each of four peptides derived by digestion of purified phosphotyrosyl p70 with trypsin is underlined. The carboxyl-terminal catalytic domain of Fpr3, which possesses peptidylprolyl cis- and trans-isomerase activity and which is homologous to other FK506- and rapamycin-binding proteins, is overlined. All of the tyrosine residues are shown as white-on-black letters. Two tyrosine residues altered by site-directed mutagenesis (Tyr-184 and Tyr-189) are marked by the asterisks. The predicted 413-residue sequence of Fpr3 that we determined and have subsequently reconfirmed (18) (GenBank accession number L34569), differs from that reported for Fpr3/Npi46 by the laboratories of Mélèse and colleagues (34) (GenBank accession number X79379) and Movva and co-workers (35) by having two additional Glu residues (codons 241 and 242).
Figure 7:
p70 is
the product of the FPR3 gene. Panel A, the presence
of phosphotyrosyl p70 is dependent upon FPR3 expression.
Wild-type strain PJ55-16A (lanes 1, 5), the ptp1 strain PJ55-16C (lanes 2, 6), the ptp1
strain YBB200 expressing the
plasmid YEp351GAL-FPR3myc (lanes 3, 7), and
the ptp1
fpr3
strain PJ55300 (lanes 4, 8) were grown in the presence of galactose. Lysates were
analyzed by immunoblotting with anti-phosphotyrosine mAb 4G10 (lanes 1-4) or polyclonal anti-Fpr3 antibody (lanes
5-8). Panel B, Ptp1 dephosphorylates Fpr3. Protein
extract from a ptp1
fpr3
strain expressing Fpr3
under the control of the GAL1 promotor
(YBB300[YEp31GAL-Fpr3]) was resolved by SDS-PAGE and
transferred to a PVDF membrane. Individual lanes were incubated at 30
°C either with buffer alone (lane 1) or with bacterially
produced Ptp1 (lanes 2-4) for the times indicated.
Levels of phosphotyrosyl Fpr3 were determined by anti-phosphotyrosine
immunoblot analysis.
To determine whether
phosphotyrosyl Fpr3 is a direct substrate of Ptp1, PVDF membrane strips
containing Fpr3 were incubated either with buffer alone or with buffer
containing 0.5 µM soluble recombinant Ptp1. Incubation in
buffer alone had no effect on the level of phosphotyrosyl Fpr3 (Fig. 7B, lane 1) nor did incubation with
Ptp1(C252A) or with Ptp1 in the presence of vanadate (data not shown).
However, incubation with Ptp1 for 1 h led to a 70% reduction in
the phosphotyrosine content of Fpr3 (Fig. 7B, lanes
2-4), as determined by densitometry. The amount of Fpr3 as
detected by immunoblotting with anti-Fpr3 antibodies was equivalent in
every lane after the incubation (data not shown). Because the substrate
protein in this reaction was immobilized and the phosphatase was
purified from bacteria (which lack PTPs), we conclude that Ptp1 can
directly dephosphorylate Fpr3. Some phosphotyrosyl-Fpr3 remained
phosphorylated following Ptp1 treatment; this may be because the
phosphotyrosine residue was inaccessible in a fraction of the
immobilized molecules.
Figure 8:
The site of tyrosine phosphorylation in
Fpr3. Panel A, Fpr3 tyrosine phosphorylation occurs within the
amino-terminal, nucleolin-like domain. The fpr3 strain
YBB100 and the ptp1
fpr3
strain YBB300 were
transformed with constructs expressing full-length FPR3
(YEp351GAL-FPR3), the amino-terminal domain (YEp351GAL-FPR3N), or the
COOH-terminal domain (YEp351GAL-FPR3C). Following galactose induction,
extracts were prepared from YBB100(YEp351GAL-FPR3) (lanes 1, 7), YBB300(YEp351GAL-FPR3) (lanes 2, 8),
YBB100(YEp351GAL-FPR3N) (lanes 3, 9),
YBB300(YEp351GAL-FPR3N) (lanes 4, 10),
YBB100(YEp351GAL-FPR3C) (lanes 5, 11), and
YBB300(YEp352GAL-FPR3C) (lanes 6, 12). The lysates
were analyzed by immunoblotting with anti-phosphotyrosine mAb 4G10 (left panel) and anti-Fpr3 polyclonal antibodies (right
panel). The samples in lanes 1-4 and 7-10 were separated on an 8.5% SDS-PAGE gel, and the samples in lanes 5, 6, 11, and 12 were
separated on a 13% SDS-PAGE gel. Panel B, Tyr residue 184 is
required for Fpr3 tyrosine phosphorylation. Protein extracts from
strain YBB300 (ptp1
fpr3
) expressing either
wild-type (wt) FPR3 or FPR3 mutated at putative
tyrosine phosphorylation sites were analyzed for relative levels of
phosphotyrosyl Fpr3 by immunoblotting with anti-phosphotyrosine or with
anti-Fpr3 antibodies. Wild-type or mutant FPR3 genes were
expressed from the YEp351GAL plasmid. Lanes 1 and 5,
wild-type FPR3; lanes 2 and 6, Y184F,Y189F double
mutant; lanes 3 and 7, Y184F single mutant; lanes
4 and 8, Y189F single mutant.
Inspection of the sequence of the amino-terminal domain of Fpr3 revealed that two of the Tyr residues (Tyr-184 and Tyr-189) are immediately preceded by two or more acidic residues (Fig. 6). This sequence context is favored by many of the Tyr-specific protein kinases in higher eukaryotes(36) . To determine if Tyr-184 and -189 were sites of tyrosine phosphorylation, these residues were changed to Phe by site-directed mutagenesis of FPR3. Neither the Y184F,Y189F double mutant nor the Y184F single mutant contained detectable phosphotyrosine (Fig. 8B, lanes 2 and 3). In contrast, the Y189F mutant possessed just as high a level of phosphotyrosine as wild-type Fpr3 (Fig. 8B, lanes 1 and 4). Thus, Tyr-184 appears to be the sole site of tyrosine phosphorylation of Fpr3.
Figure 9:
Fpr3 is phosphorylated at Ser, Thr, and
Tyr in vivo and in vitro. Panels 1 and 2, phosphoamino acid analysis of Fpr3 labeled in
vivo. Strains YPH499 (PTP1) and YBB200 (ptp1) expressing YEp351-FPR3myc were
metabolically labeled with
P
for 3 h,
harvested, and disrupted by alkaline lysis. Fpr3 was immunoprecipitated
with rabbit anti-Fpr3 antibody and subjected to phosphoamino acid
analysis. Autoradiography was carried out by exposure for 48 h in a
Phosphorimager. Panel 3, phosphorylation of GST-Fpr3N in
vitro. GST-Fpr3N adsorbed to glutathione-Sepharose beads was
incubated with lysate from the protease-deficient ptp1
strain YLW200, washed, and then incubated in the presence of
[
-
P]ATP. The GST-Fpr3N was resolved by
SDS-PAGE and subjected to phosphoamino acid analysis. Autoradiography
was carried out by exposure for 24 h to x-ray film with an intensifying
screen. In all three panels, position of phosphoamino acids detected by
ninhydrin staining is marked by S (phosphoserine), T (phosphothreonine), or Y (phosphotyrosine).
The broad substrate
specificity of Ptp1 suggests several possible functions for the enzyme.
One extreme possibility is that Ptp1 may totally lack specificity for
protein substrates, and function simply to reverse adventitious
tyrosine phosphorylation by error-prone or promiscuous Tyr-specific or
dual-specific protein kinases. Consistent with this idea, we observed
that ptp1 yeast were killed by mutants of the v-src tyrosine kinase that were only partially growth inhibitory in PTP1 strains. (
)However, other observations suggest
that Ptp1 has some level of substrate specificity and thus that it may
have a more specific role in yeast cell physiology. It is clear that
Ptp1 is unable to fulfill the functional niches occupied by other PTPs
in S. cerevisiae. The fact that cells carrying mutations in a
PTP-encoding gene, CDC14, undergo a cell cycle arrest (9) is evidence that, under normal conditions, Ptp1 cannot
dephosphorylate the substrate(s) of Cdc14. Recent genetic evidence
indicates that Ptp2 may function by dephosphorylating Hog1, the
terminal MAP kinase of the osmosensory signaling pathway. The SLN1 gene encodes a histidine-protein kinase receptor that mediates
this osmosensory pathway. Overexpression of PTP2 (but not of PTP1) rescues sln1 mutants, and normal expression of PTP2 (but not of PTP1) can compensate for a mutation
in the functionally related phosphatase, Ptc1(8, 15) .
It will be of interest to determine whether the limitations on the
activity of Ptp1 are a result of its subcellular localization (see
below) or an inability to recognize and dephosphorylate certain
phosphotyrosyl proteins.
At present, we do not know whether tyrosine phosphorylation affects Fpr3 function. Indeed, the precise cellular function of Fpr3 is unknown, but the properties of related mammalian and yeast immunophilins provide several clues. The peptidyl-prolyl isomerase activity of immunophilins suggests that they may catalyze protein folding (reviewed in (38) ). The immunosuppressant drugs, FK506 and rapamycin, which mimic the peptidyl-prolyl bond, bind to the FKBP class of immunophilins. In mammalian T-cells, the complex of drug and immunophilin blocks signal transduction. In yeast, exposure to FK506 inhibits calcineurin-mediated signal transduction and certain amino acid permeases(39) , while exposure to rapamycin is lethal(40) . Three FKBPs have been described in S. cerevisiae: Fpr1, Fpr2, and Fpr3. Fpr1, a homolog of the mammalian FKBP-12, is a cytosolic protein with high affinity for FK506 and rapamycin. FPR1-deficient yeast are resistant to these drugs, indicating that Fpr1 is largely responsible for mediating drug toxicity(41, 42, 43) . S. cerevisiae FPR2, a homolog of mammalian FKBP-13, may be involved in the proper folding of proteins in the ER(44, 45) .
Fpr3
is an abundant nucleolar protein that is dispensable for growth. Yeast
with disruptions in FPR3, including fpr1 fpr2 fpr3 strains, grow normally under a variety of growth conditions. The
drug-binding and proline isomerase activities of Fpr3 are mediated by
the conserved immunophilin domain, which represents the
carboxyl-terminal third of Fpr3. When this domain is expressed
independently, it is retained in the cytoplasm and restores FK506 and
rapamycin sensitivity in fpr1 strains. The amino-terminal
two-thirds of Fpr3 contains striking regions of acidic and basic
residues and is responsible for localization of Fpr3 in the nucleolus (18, 34, 35) . This portion of Fpr3 exhibits
some sequence similarity to nucleolin, a major nucleolar protein
thought to be involved in ribosome assembly and shuttling of RNA or
proteins through nuclear pores (for review, see (46) ).
Our
findings also indicate that Fpr3 is tyrosine phosphorylated within the
amino-terminal nucleolar localization domain. Preliminary
immunofluorescence studies suggest that Ptp1 is localized primarily in
the cytosol. ()These observations raise the possibility that
Fpr3 might be dephosphorylated by Ptp1 prior to its entry into the
nucleus and that tyrosine phosphorylation and dephosphorylation of Fpr3
might regulate its subcellular localization. By dephosphorylating Fpr3,
Ptp1 might also play a role in regulating the catalytic activity of
Fpr3 and/or the ability of Fpr3 to associate with other proteins in the
nucleolus. The Y184F mutant of Fpr3 does not undergo tyrosine
phosphorylation and will serve as a useful reagent to study the
possible effects of tyrosine phosphorylation/dephosphorylation on Fpr3
localization and catalytic activity.
Because dedicated tyrosine-specific protein kinases have not been identified in unicellular eukaryotes, it is possible that a dual specificity kinase is responsible for the phosphorylation of Fpr3 at Tyr-184. Alternatively, the Fpr3 tyrosine kinase might be a novel yeast kinase that is tyrosine-specific. We have shown here that yeast extracts contain an activity or activities that phosphorylate Fpr3 at tyrosine, as well as at serine and threonine. Identification of the Fpr3 tyrosine kinase should allow us to distinguish between these possibilities.