(Received for publication, January 27, 1995; and in revised form, June 30, 1995)
From the
We have identified a mouse gene encoding a 65-kDa protein
(FKBP65) that shares homology with members of the FK506-binding protein
(FKBP) class of immunophilins. Predicted amino acid sequence shows that
this protein shares significant homology with FKBP12 (46%), FKBP13
(43%), FKBP25 (35%), and FKBP52 (26%). FKBP65 contains four predicted
peptidylprolyl cis-trans-isomerase (PPIase) signature domains,
and, although similar in size, is distinct from FKBP52 (also identified
as FKBP59, hsp56, or HBI), which contains three FKBP12-like PPIase
domains. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the
substrate, recombinant FKBP65 is shown to accelerate the isomerization
of the prolyl peptide bond with a catalytic efficiency similar to other
family members. This isomerization activity is inhibited by FK506 and
rapamycin, but is not sensitive to Cyclosporin A. Based on Northern
blot analysis, FKBP65 mRNA transcripts are present in lung, spleen,
heart, brain, and testis. A polyclonal antibody, raised against a
COOH-terminal peptide (amino acid residues 566-581), was used to
immunoprecipitate FKBP65 from NIH3T3 cells and demonstrate that FKBP65
is a glycoprotein. In addition,
[P]orthophosphate labeling experiments show that
FKBP65 is also a phosphoprotein. These results suggest that FKBP65 is a
new FKBP family member.
FK506 and rapamycin are immunosuppressant macrolide antibiotics
that mediate their activity in part by binding to members of a
ubiquitous family of highly conserved intracellular receptors termed
immunophilins(1, 2) . Although FK506 and rapamycin are
structurally related and bind to a distinct class of immunophilins
termed FK506-binding proteins (FKBPs), ()they exert their
immunosuppressive effects by blocking different signal transduction
pathways in normal T cells. FK506 selectively inhibits the
Ca
-dependent signal transduction events by
specifically binding calcineurin, a Ca
- and
calmodulin-dependent serine/threonine phosphatase(3) .
Inhibition of calcineurin phosphatase activity interferes with an early
event that is essential for T-lymphocyte growth and
differentiation(4, 5) . In comparison, rapamycin
appears to affect Ca
-independent, IL-2-driven T-cell
proliferation(6, 7) . A characteristic shared by
immunophilins is peptidylprolyl cis-trans-isomerase (PPIase)
activity(8, 9) , which is inhibited upon drug
binding(10) . However, the inhibition of PPIase activity in
itself does not appear to be responsible for the immunosuppressant
effect of the drugs(7, 11) .
To date, four FKBP isoforms, named according to their calculated molecular mass: FKBP12(8, 10) , FKBP13(12) , FKBP25(13, 14, 15) , and FKBP52(16, 17) , have been identified.
This report describes the molecular cloning, sequencing, and biochemical characterization of a novel 65-kDa protein, FKBP65, isolated from a NIH3T3 fibroblast cDNA library. We show that this protein, as well as containing four peptidylprolyl cis-trans-isomerase domains, possesses PPIase activity comparable to other FKBPs and appears to be a new and distinct FKBP family member.
Figure 1:
Analysis of nucleotide and predicted
amino acid sequence of murine FKBP65. A, numbers on the left indicate nucleotide positions, and numbers on the right indicate amino acid residues. The 2.6-kb clone contains
a 1743-bp open reading frame (nucleotides 94-1837), and a
polyadenylation signal (AATGAAA) is located at bp 2431-2437. The bold sequence is the predicted NH-terminal signal
sequence. The italicized sequence is a potential
membrane-spanning sequence. The underlined sequence was used
to generate anti-peptide 4 (Pep 4) polyclonal antiserum. B,
alignment of the amino acid sequence of the four FKBP65 PPIase domains
with the central region of hFKBP12. Residues conserved in at least
three sequences are boxed. The seven residues considered
important for FK506 binding are denoted with an
*.
The
predicted amino acid sequence of FKBP65 revealed a highly hydrophobic
region (residues 14-23) suggesting an NH-terminal
signal sequence (Fig. 1A, bold sequence). We
analyzed the amino acid pattern of FKBP65 using the method of Von
Heijne (27) for predicting potential cleavage processing sites
in a pre-protein. Based on this method, we predict that FKBP65 is
cleaved after Arg
, resulting in a mature protein with a
calculated molecular mass of 60,576 daltons. Additionally, there is a
potential membrane-spanning segment (amino acid residues 244-258)
consisting of hydrophobic residues (Fig. 1A, italicized sequence), as well as an ER retention sequence
(HEEL) at the COOH terminus(28) .
Analysis of the 2.6-kb clone, using the University of Wisconsin GCG software analysis program(29) , revealed a domain containing significant amino acid sequence similarity to members of the FK506/rapamycin-binding protein (FKBP) family(19) . In accordance with the convention for naming FKBP family members corresponding to their calculated molecular weights(17) , we have renamed the protein previously designated FKBPRP (19) as FKBP65. Overall, FKBP65 shares 46% and 43% amino acid sequence identity with human FKBP12 and FKBP13, respectively, and 35% and 26% sequence homology with human FKBP25 and FKBP52 (FKBP59, hsp56, or HBI).
Additional sequence analysis of the 2.6-kb FKBP65 clone revealed four peptidylprolyl cis-trans-isomerase (PPIase) signature domains as defined by the Motifs program (GCG, University of Wisconsin, Madison, WI; (29) ). These four domains are located at amino acid residues 61-149, 173-261, 285-373, and 398-485 and represent 67% of the protein (Fig. 1B). The regions flanking the four PPIase domains share 30% similarity; however, they appear to be unique as they show no homology with other FKBP family members or any sequences reported in available data bases. The FKBP65 PPIase Domain II conforms to the PPIase consensus motif, whereas the other three domains each contain one mismatch (Fig. 1B).
Crystallographic studies of FK506 bound
to human FKBP12 (hFKBP12) have identified two regions within the PPIase
domain that appear to be important for FK506-binding interactions.
There are five amino acid residues (Tyr, Phe
,
Val
, Ile
, and Trp
) proposed to
form the hydrophobic drug binding cavity and three amino acid residues
(Ile
, Asp
, and Tyr
) of hFKBP12
that appear to form hydrogen bonds with
FK506(30, 31) . These seven amino acids are conserved
in all FKBPs that have been identified to date. Sequence alignment of
hFKBP12 with the four PPIase domains of FKBP65 reveals that Domains
I-III of FKBP65 strictly conserve six of these seven amino acid
residues and Domain IV conserves five of the seven amino acids (Fig. 1B).
Unlike the ubiquitously expressed FKBPs identified to date, FKBP65 mRNA shows restricted expression. Northern blot analysis reveals a unique 2.6-kb RNA band in mouse lung, spleen, heart, and brain that hybridizes to a radiolabeled 1.7-kb (ORF) fragment of the FKBP65 clone (Fig. 2). The testis mRNA appears to contain two transcripts, the 2.6-kb band and an additional 3.5-kb band. Although RNA was visualized in all lanes, by either ethidium bromide staining of the gel prior to blotting or hybridization with an actin probe (data not shown), no FKBP65-specific hybridization in liver RNA was seen.
Figure 2: Northern blot analysis of FKBP65 in mouse tissues. Total RNA (20 µg) from various mouse tissues were electrophoresed in a 1% agarose gel, blotted to nylon membranes, and hybridized with the 1.7-kb ORF cDNA probe.
Figure 3: Western blot analysis of recombinant FKBP65 purification and inhibition of FKBP65 isomerase activity. A, Western blot analysis of rFKBP65 before (lane 1) and after (lane 2) RP-HPLC purification as described under ``Experimental Procedures.'' The smaller apparent size of rFKBP65 is due in part to improper processing of the protein in the bacterial cell. B, progress curves of the chymotrypsin-coupled assay of PPIase activity using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (108 µM) as the peptide substrate with (curve a) or without (curve e) purified rFKBP65 (16 nM). The PPIase activity of purified rFKBP65 is inhibited in a dose-dependent manner by the addition of 27 (curve b), 54 (curve c), or 200 nM (curve d) FK506.
To detect FKBP65
protein, two polyclonal antisera (Pep 2 and Pep 4) were raised in
rabbits against synthetic peptides derived from the predicted sequence
of FKBP65, as described under ``Experimental Procedures.''
Although both peptide antisera gave similar results, we used Pep 4
antiserum, raised against the carboxyl terminus of FKBP65 (Fig. 1A, underlined sequence) because it
reacted more strongly in immune precipitations and Western blots. Pep 4
antiserum was used to immunoprecipitate total cell lysates of
[S]methionine-labeled NIH3T3 cells. Despite the
calculated molecular mass of 64,683 or 60,576 for the
NH
-terminal cleaved protein, two prominent bands migrating
at approximately 72 and 68 kDa were observed (Fig. 4, lane
2) that were specifically competed when peptide was added during
immune precipitation (Fig. 4, lane 3). The discrepancy
between apparent and calculated molecular masses is observed with other
FKBP family
members(9, 10, 12, 14, 16, 33) .
Figure 4:
Identification of FKBP65 in
[S]methionine-labeled murine NIH3T3 epidermal
cells. A, immunoprecipitation with Pep 4 antiserum of NIH3T3
total cell lysates. Two bands that migrate at 72 and 68 kDa,
respectively, were specifically precipitated by Pep 4 antiserum (lane 2), but not by preimmune antiserum (lane 1) or
with Pep 4 antiserum in the presence of competing peptide (lane
3). B, endoglycosidase F (Endo-F) treatment of
[
S]methionine-labeled NIH3T3 lysates. Endo-F
treatment (+) of Pep 4 antiserum precipitates reveals a single
specific band that migrates at an apparent molecular mass of 58 kDa (lane 4). Cell lysates were precipitated with protein
A-Sepharose coupled to either preimmune (lane 1) or with
anti-peptide 4 antiserum in the absence (lanes 2 and 4) or presence of competing peptide (lanes 3 and 5) as described under ``Experimental
Procedures.''
The increase in apparent molecular weight may be due to the presence of post-translational modifications such as glycosylation and/or phosphorylation. The predicted amino acid sequence contains seven potential N-glycosylation sites and 14 potential casein kinase II phosphorylation sites. Additionally, FKBP65 contains one cAMP-dependent protein kinase phosphorylation site, one protein kinase C phosphorylation site, and two tyrosine kinase phosphorylation sites. Furthermore, FKBP65 contains nine proline-proline sequences that may alter the protein structure resulting in an altered migration on SDS-PAGE and increasing its apparent size.
To determine if FKBP65
was glycosylated, NIH3T3 cells were metabolically labeled with
[S]methionine, precipitated with Pep 4
antiserum, and incubated for 3 h with N-endoglycosidase F
(Endo-F; Fig. 4, lanes 4 and 5) or in Endo-F
buffer alone (Fig. 4, lanes 1-3). The
Endo-F-treated FKBP65 migrates at an apparent molecular weight of
58,000 (Fig. 4, lane 4). To confirm that the 58-kDa
band was the unglycosylated form of FKBP65, Western blot analysis of
Endo-F-treated lysates was performed (data not shown), and an
immunoreactive 58-kDa band was visualized. Additionally, when NIH3T3
cells are pretreated with either 2-deoxyglucose or tunicamycin, agents
that interfere with protein glycosylation, the unglycosylated 58-kDa
species also was observed. These results demonstrate that FKBP65 is a
glycoprotein.
The observance of two precipitated bands may reflect
the difference between phosphorylated and unphosphorylated forms of
FKBP65 or alternatively may represent the product from a different
translational start site. There is a second methionine residue 60 amino
acids downstream from the first ATG sequence. This second ATG contains
a nearly perfect Kozak consensus sequence (26) and may encode a
protein with a molecular weight of approximately 57,000; however,
typically only a single 2.6-kb transcript is seen (Fig. 2). To
determine if FKBP65 also is phosphorylated, NIH3T3 cells were labeled
with [P]orthophosphate followed by immune
precipitation with Pep 4 antiserum as described under
``Experimental Procedures.'' The immune precipitates were
visualized by autoradiography after SDS-PAGE and electroblotting onto
ImmobilonP
. A specific band at a relative molecular mass
of 72,000 (Fig. 5A, lane 2) was observed. The
P-labeled blot was allowed to decay prior to Western blot
analysis (Fig. 5B), and alignment of the two
autoradiographs confirms that the
P-labeled protein is
FKBP65 and represents the top 72-kDa band observed during immune
precipitation.
Figure 5:
FKBP65 is a phosphoprotein. A,
total cellular lysates of NIH3T3 cells labeled with
[P]orthophosphate were precipitated with either
preimmune antiserum (lane 1) coupled to protein A-Sepharose or
with Pep 4 antiserum in the absence (lane 2) or presence of
competing peptide (lane 3), separated on SDS-PAGE, and
electroblotted onto ImmobilonP
as described under
``Experimental Procedures.'' B, Western blot
analysis of the filter in A.
We have identified a mouse gene encoding a 65-kDa protein
(FKBP65) that shares homology with members of the FK506-binding protein
class of immunophilins. In this study, we report the cloning,
sequencing, and biochemical characterization of a unique 2636-bp clone
isolated from a NIH3T3 fibroblast Zap cDNA library. The 2.6-kb
FKBP65 clone contains an ORF encoding a protein with a predicted
molecular mass of 64,683 daltons; however, the presence of a
hydrophobic leader sequence and a predicted cleavage site would result
in a mature protein with a molecular mass of 60,576. Amino acid
sequence comparison of FKBP65 to other FKBP family members revealed
from 26% (human FKBP52) to 46% (hFKBP12) overall sequence homology.
Additionally, FKBP65 contains four FK506-binding protein PPIase
signature domains. The regions flanking the four PPIase domains retain
30% similarity and appear to be unique as they are not homologous to
other FKBPs or any sequences reported in available data bases. The
first three domains of FKBP65 conserve six of the seven amino acid
residues reported to be involved with FK506-binding interactions.
We
have expressed rFKBP65 in the pET21d expression vector
and demonstrate that rFKBP65 has PPIase activity with kinetics similar
to other FKBPs that is inhibitable by FK506 and rapamycin, but not
Cyclosporin A. Although FKBP65 is approximately the same relative size
of the larger, multidomain FKBP52 that associates with steroid
receptors(16, 17, 33, 34, 35) ,
FKBP65 is distinct from FKBP52 that contains three FKBP12-like PPIase
domains. Likewise, FKBP65 appears to be distinct from p50 and p54, two
FKBP-related proteins recently shown to complex with the avian
progesterone receptor(36, 37) . Collectively, these
observations strongly suggest that FKBP65 is a new FKBP family member.
Another distinguishing factor of FKBP65 is the tissue distribution.
A unique 2.6-kb mRNA band was found in lung, spleen, heart, and brain,
but not in liver. This result suggests that FKBP65, unlike other FKBP
family members, may not be ubiquitously expressed. The second, larger
band detected in the testis may reflect a related protein or an
alternate form of FKBP65. We have recently isolated a partial human
clone ()that, when used as a probe, detects a 3.5-kb band in
human mRNA. Using a rabbit polyclonal peptide antibody, two specific
bands (72 and 68 kDa) were detected by immunoprecipitation and Western
blot analysis in NIH3T3 cell lysates. The appearance of two
precipitable bands may be the product resulting from a second,
downstream methionine or reflect a difference in phosphorylation
states. When cells were labeled with
[
P]orthophosphate, only the upper, 72-kDa band
was visualized; however, both bands are observed on Western blot
analysis. Although FKBP65 appears to be a phosphoprotein, there is no
identified ATP binding site within the sequence. We have shown that
FKBP65 is a glycoprotein, an observation that can account for the
difference observed between the calculated and apparent molecular
weights; however, the unglycosylated form migrates at an apparent
molecular weight of 58,000 that is smaller than the predicated
molecular weight of 60,576. This additional difference may be due in
part to the large number of proline residues (37 in the mature protein)
including nine Pro-Pro sequences that may alter protein structure
leading to an altered migration on SDS-PAGE.
The functional diversity of the FKBP family members may be due to their distinct subcellular localization and association with different protein complexes. The multidomain FKBP52 has been shown to associate with hsp90, hsp70, and the unactivated steroid receptors(16, 17, 33) , whereas the unidomain FKBP25, which is localized in the nucleus, associates with casein kinase II and nucleolin(38) . The smaller FKBPs, FKBP12 and FKBP13, have been localized to the cytoplasm (8) and the endoplasmic reticulum(39) , respectively. FKBP12 has been shown to associate with the ryanodine receptor(40) , and, recently, the inositol 1,4,5-trisphosphate receptor(41) , both of which mediate calcium release processes. The predicted amino acid sequence of FKBP65 reveals a potential ER retention sequence and a possible membrane-spanning sequence, suggesting localization to the ER. Using various cellular fractionation protocols, we have localized FKBP65 to the membrane and cytosolic fractions; however, due to the presence of N-linked oligosaccharides, the appearance of FKBP65 in the cytosolic fraction may be a result of inadequate fractionation and/or contamination from the ER fractions.
The presence of four PPIase
domains of FKBP65 suggest that they may function to interact with more
than one molecule within a complex of proteins or act
stoichiometrically with the same molecule. We are currently
investigating proteins that associate with FKBP65, using
affinity-purified protein and NIH3T3 lysates with or without FK506 or
rapamycin treatment. To identify the possible functions of FKBP65 and
associated proteins, we are currently generating FKBP65 mutants to
assess the functional roles of the four PPIase domains. Using the Pep 4
polyclonal antibody, immunoreactive FKBP65 also has been detected by
immunoprecipitation and Western blotting techniques in human cell
lines, and we are currently in the process of isolating the
human homologue of FKBP65. This will allow precise comparisons of the
various human FK506-binding proteins and possible detection of
additional genes related to FKBP65.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L07063[GenBank].