ABBS 2005,38(03): Analysis of the Interaction between hPFTAIRE1 and PLZF in a Yeast Two-hybrid System

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Abstract        hPFTAIRE1 is
a Cdc2-related kinase family member. To search its substrates and regulatory
proteins, hPFTAIRE1 was fused to LexA and used as a bait to screen a human
brain LexA two-hybrid library. In this screening, seven hPFTAIRE1 interacting
proteins, including promyelocytic leukemia zinc finger (PLZF), were obtained.
The interaction between PLZF and hPFTAIRE1 was confirmed by b-galactosidase assay and Leu growth activity. PLZF
encodes a transcription factor belonging to the POZ/BTB domain and Krüppel zinc
finger (POK) family. The highly conserved POZ/BTB domain plays a critical role
in protein-protein interaction. We deleted the POZ/BTB and Krüppel zinc finger
domains, respectively, and observed the interaction between hPFTAIRE1 and
truncated PLZFs by liquid b-galactosidase
activity assay. A weak interaction was detected between hPFTAIRE1 and PLZF. We
also observed the interaction between PLZF and another Cdc2-related kinase,
PCTAIRE1. A similar result was observed. The interaction between PLZF and
hPFTAIRE1 or PCTAIRE1 was confirmed by co-immunoprecipitation assay in a yeast
system. PLZF is a phosphoprotein and plays multiple roles during cell growth.
Our results suggest that hPFTAIRE1 and PCTAIRE1 may play important roles in the
functional regulation of PLZF.

Key words     hPFTAIRE1; PLZF; PCTIARE1; yeast
two-hybrid; interaction

Cyclin-dependent kinases
(CDKs) are serine/threonine kinases that have been shown to be key players in
the control of cell cycle progression [1]. These various CDKs share high
similarity in their amino acid sequence, and contain a PSTAIRE motif for
binding of their cyclin partners. Over the last few years, a number of kinases
have been identified in mammalians based on structural similarity with p34cdc2 [2], but their cyclin partners remain unidentified. These
Cdc2-related protein kinases derived their names from the presence of an amino
acid for serine substitution in the cyclin binding sequence PSTAIRE. Some of
these Cdc2-related kinases, such as PFTAIRE, PCTAIRE, PITSIRE and Cdk5, have
been shown to play important roles during cell division, gametogenesis and
differentiation [3–6]. Two mouse
PFTAIRE (PFTAIRE and mPFTAIRE1) genes have been reported
[7,8]. mPFTAIRE1 has a 46
amino acid extension compared to PFTAIRE at its N-terminus. PFTAIRE is expressed primarily in the
postnatal and adult nervous system by in situ hybridization and indirect
immunofluorescence. In neurons, the PFTAIRE protein was localized in the
nucleus and cytoplasm of cell bodies. mPFTAIRE1 is ubiquitously
expressed in murine tissues and highly expressed in brain, testis and embryo.
mPFTAIRE1 is implicated in the process of meiosis as well as neuron
differentiation. We have isolated a Cdc2-related protein kinase, PFTAIRE1, from
humans (hPFTAIRE1) [9]. hPFTAIRE1 was highly expressed in brain,
pancreas, kidney, heart, testis and ovary. To search for its substrate, we
screened an adult human brain two-hybrid cDNA library using hPFTAIRE1 as bait
and identified several hPFTAIRE1 interacting proteins, including septin family
members KIAA0202 [10] and PLZF.

The PLZF gene was
initially identified by virtue of its fusion with RARa as a result of a variant (11;17) chromosomal
translocation that occurs in a small subset of acute promyelocytic leukemia
(APL) patients [11,12]. The PLZF protein, highly conserved among humans, mice
and chickens [13], is a nuclear protein containing nine Krüppel-type zinc
finger domains at the carboxyl terminus, which bind directly to DNA [14]. The
amino terminus of PLZF contains a BTB/POZ domain that mediates
self-dimerization and transcriptional repression through binding to nuclear
corepressors [15]. The target genes of PLZF include cyclin A and the
interleukin (IL)-3 receptor alpha chain [16]. PLZF overexpression in
hematopoietic cell lines leads to growth suppression, apoptosis, G1/S phase
cell cycle arrest, and differentiation blockade [17]. Suppression of cyclin A
expression by PLZF may contribute to the growth suppressive properties of PLZF
[16]. Analysis of PLZF knockout mice demonstrate that PLZF is largely
implicated in development, especially in limb and axial skeleton patterning,
and Hox gene regulation in vivo [18]. The PLZF protein was postulated to
play a specific role in early hematopoiesis [17]. PLZF is a negative regulator
of cell cycle progression, ultimately leading to growth suppression.

In this study, we
screened hPFTAIRE1 interacting proteins in a two-hybrid system and identified
PLZF. The interaction between hPFTAIRE1 and PLZF was analyzed. We also analyzed
the interaction between PLZF and another CDK family member, PCTAIRE1.

Materials and Methods

Strains and growth
conditions

The Escherichia coli
strain used in this study was DH5a (supE44DlacU169 hsdR17
recA1 endA1 gyrA96 thi-1 relA1) [24]. The Saccharomyces
cerevisiae strain used in this study was CJY151 (MATa, his3, trp1, LexAop(´6)-leu2, LexAop(´8)-lacZ), which was
generated by integrating the LacZ reporter and its upstream modulation sequence
to the strain EGY48 (MATa, trp1
ura3, his3, leu2, LexAop((6)-leu2). E. coli
was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) at 37
ºC. S. cerevisiae strains were cultured in YPD medium (2% tryptone, 1%
yeast extract, 2% glucose) at 30 ºC or SC/Glu or SC/Gal at 30 ºC.

Plasmid construction

The full open reading
frame of the hPFTAIRE1 gene was inserted into the EcoRI and BamHI
sites of the pGilda vector to generate pGilda-PFT, which was used as a bait
plasmid for two-hybrid screening. The full open reading frame of the PCTAIRE1
coding region was inserted into the EcoRI and BamHI sites of the
pGilda vector to generate pGilda-PCT. Full-length PLZF was cloned into
the EcoRI and XhoI sites of pB42AD to construct pB42AD-PLZF. All
the constructed plasmids were confirmed by DNA sequencing.

Yeast two-hybrid
screening

A human adult brain LexA
two-hybrid cDNA library (Clontech, The pGilda-PFT and
library plasmids were sequentially transformed into yeast host strain CJY151 (MATa, his3, trp1, LexAop(´6)-leu2, LexAop(´8)-lacZ) [19]. A
total of 1´108 transformants were plated onto the yeast
galactose drop-out minimal medium lacking histidine, tryptophan and leucine,
and incubated for 3 d at 30 ºC. The Leu+ colonies
were collected and assayed for b-galactosidase
activity. The library plasmids from the positive colonies (Leu+ and b-Gal+) were confirmed by
polymerase chain reaction (PCR) based restriction map and DNA sequence
analysis.

Mutagenesis and b-galactosidase
activity assay

Three PLZF mutants, PLZFDBTB (PLZF with BTB domain deleted), PLZFDzinc finger (PLZF with zinc finger domain deleted)
and PLZFDN (PLZF with
only zinc finger domain available), were constructed into the EcoRI and XhoI
sites of pB42AD by PCR amplification using the human full-length PLZF as
a template. All plasmids were sequenced for correct construction. Various pairs
of the pGilda and pB42AD plasmids were co-transformed into yeast reporter
strain CJY151 (MATa, his3,
trp1, LexAop(´6)-leu2, LexAop(´8)-lacZ) [19] to
test for b-galactosidase
activity. b-galactosidase
assays with ONPG and X-gal as substrates, respectively, were performed, as
described previously (MATCHMAKER two-hybrid system, Clontech).

Co-immunoprecipitation
assay

Approximately 2 mg of
total protein extracts was used for each co-immunoprecipitation assay. Each
sample was incubated with 3 mg antibody ( 

Results

hPFTAIRE1 used as bait
to screen interacting proteins

The protein kinase
hPFTAIRE1 was highly expressed in human brain [9]. Therefore an adult human
brain LexA two-hybrid library was chosen to search for its putative substrates.
A full-length hPFTAIRE1 was fused with LexA and inserted into the pGilda
vector to generate pGilda-PFT for expression of LexA-hPFTAIRE1 protein (Fig.
1). pGilda-PFT, co-transformed with control vector, had no detectable b-galactosidase activity [Fig. 2(A)].
Results indicated that the hPFTAIRE1 protein could be used as bait for
two-hybrid screening.

Identification of PLZF
from screening

pGilda-PFT was
co-transformed with the two-hybrid library in yeast strain CJY151. Among 1108 colonies screened, 243 colonies could grow on
the minimal medium (SC Gal/Raff ura– his– trp– leu–), and 81 of
these showed a positive response in the b-galactosidase assay. Restriction map and DNA sequence
analysis demonstrated one of the inserts encoding for PLZF. The interaction
between hPFTAIRE1 and PLZF was confirmed by b-galactosidase assay and Leu growth activity [Fig.
2(B)]. From the screening,
we totally isolated 16 inserts, representing five kinds of fragments of PLZF (Fig.
3).

Test of the autonomous
activating activity of PLZF and its mutants

To verify the
interaction between hPFTAIRE1 and PLZF in yeast, we transformed pB42AD-PLZF
with control vector. We found that PLZF had an autonomous activating activity.

From primary structure
analysis, PLZF contains two conserved domains. At its N-terminus, PLZF contains
a BTB/POZ domain and a nine Krüppel-type zinc finger domain located at its
C-terminus (amino acids 403–673). To
determine which domain is crucial for autonomous activation, three PLZF
deletion fragments (PLZFDBTB, PLZFDzinc finger and PLZFDN) were inserted into pB42AD to express HA fusion
proteins (Fig. 4). The three mutants were each co-transformed with
pGilda into yeast strain CJY151, and wild-type PLZF was used as a control.
Liquid b-galactosidase
activity assays were performed. As shown in Fig. 4, the C-terminal zinc
finger domain is crucial for autonomous activation. The central region of PLZF
plays a minor role and deletion of the BTB/POZ domain contributes little to
autonomous activation.

Interaction between
hPFTAIRE1 and PLZF in a yeast system

Because PLZF has an
autonomous activating activity in a two-hybrid system, we verified the
interaction between hPFTAIRE1 and PLZF again. Full-length PLZF and three
mutants were co-transformed with pGilda-PFT into yeast strain CJY151. b-galactosidase activity was assayed in liquid
using ONPG as a substrate. The results are shown in Table 1. b-galactosidase activity in yeast strain containing
pGilda-PFT and pB42AD-PLZF was 7–8-fold higher
than that containing pB42AD-PLZF alone. Similar to full-length PLZF, hPFTAIRE1
showed a higher reporter activity when interacting with the PLZF mutants (Table
1). Results showed that the whole region of PLZF contributed to the
interaction and the C-terminal zinc finger domain played an important role,
both in the interaction and its autonomous activation, as deletion of this
region abolished all b-galactosidase
activity.

PCTAIRE1 is another
Cdc2-related protein kinase and shares the highest similarity with hPFTAIRE1.
More interestingly, these two kinases are highly expressed in post mitotic
cells. We also analyzed the interaction between PCTAIRE1 and PLZF. PLZF and
PLZF mutants were co-transformed with pGilda-PCT, respectively, and assayed in
liquid for b-galactosidase
activity. Results showed that PCTAIRE1 could interact with PLZF in a similar
pattern (Table 1).

To confirm the direct
interaction between hPFTAIRE1 or PCTAIRE1 and PLZF, we performed a
co-immunoprecipitation experiment in yeast cells. LexA-hPFTAIRE1 or
LexA-PCTAIRE1 was co-expressed with HA-PLZF proteins in CJY151. The yeast
lysates were immunoprecipitated by anti-HA antibody and then detected with
anti-LexA antibody using Western blot. A 77 kDa band that corresponds to
hPFTAIRE1 was detected in the pGilda-PFT and pB42AD-PLZF co-transformed cells,
and the control showed a negative result [Fig. 5(A)]. A 79 kDa band that corresponds to
PCTAIRE1 was detected in the pGilda-PCT and pB42AD-PLZF co-transformed cells,
and the control showed a negative result [Fig. 5(A)]. That the two Cdc2-related protein
kinases, hPFTAIRE1 and PCTAIRE1, could interact with PLZF suggested that
hPFTAIRE1 and PCTAIRE1 might play important roles in the functional regulation
of PLZF.

Discussion

CDKs are a family of
serine/threonine kinases, which play well-established roles in the regulation
of the eukaryotic cell division cycle and have also been implicated in the
control of gene transcription and other processes. hPFTAIRE1 is a member of the
CDK family whose function is unknown. PCTAIRE1 also belongs to the Cdc2-related
protein kinases [20], and shares high similarity with human PFTAIRE, especially
in the kinase conserved subdomains. In vitro translated PFTAIRE or
bacteria produced GST-PFTAIRE do not have kinase activity on their own protein
(autophosphorylation) or other known substrates, including histone H1 and MBP.
Regulatory components are believed to be required for its kinase activity. The
activity and function of CDKs are governed by their regulatory subunits and
phosphorylation events. We have identified several hPFTAIRE1 interacting
proteins during two-hybrid screening using full-length hPFTAIRE1 as bait. In
this study, we reported that PLZF was able to interact with two Cdc-2 related
protein kinases, hPFTAIRE1 and PCTAIRE1, suggesting that a novel regulatory
function of PLZF may be involved.

The PLZF transcriptional
repressor when fused to retinoic acid receptor alpha (RARa) causes a refractory form of acute promyelocytic
leukemia [21]. It was reported that PLZF could be phosphorylated on serine and
threonine residues and contains two potential Cdc2 phosphorylation consensus
sequences found in the region between the POZ domain and the zinc fingers of
PLZF [26]. Some researchers found that in vitro expressed zinc finger
domains bind to a much wider variety of sequences than full-length PLZF [26].
So phosphorylation of the zinc finger domain or the upstream sequence must play
an important role in PLZF DNA binding. Conversely, the substrates for hPFTAIRE1
and PCTAIRE1 kinases have not been identified. The kinase activity in the
PFTAIRE immune complex, precipitated from a mouse brain cytosolic protein
preparation, phosphorylated two specific sets of proteins approximately 58–60 kDa and 200–205 kDa in size. These proteins remained associated with
PFTAIRE during immunoprecipitation [7]. The immunoprecipitated PFTAIRE was not
able to phosphorylate histone H1, NF-H and other common substrates, suggesting
that PFTAIRE may have a more restricted set of substrates than other CDKs. PLZF
is a phosphoprotein, but its specific upstream phosphorylating kinases have not
been reported yet. Our study suggests that hPFTAIRE1 and PCTAIRE1 could
interact with PLZF, mainly through linker region and zinc finger domains. In
the linker region, there is one domain termed the proline-rich domain (Pro
domain) [14,27] or second repression domain (RD2) that is critical in forming a
complex with ETO [28].

Recent studies
identified PLZF as a new androgen upregulated gene in the prostate
[22,23] and suggested that PLZF might play important roles in androgen
action. It was also reported that PLZF might function in adult germline stem
cell self-renewal. The PCTAIRE1 kinase has been reported to have a role in
spermatogenic cells because its kinase activity was only detected in those
cells [5]. It has been suggested that mouse PFTAIRE may have a role in the
process of meiosis [8]. Cdc2-related protein kinase plays an important role in
the regulation of gene transcription. Interaction between PLZF and two kinases
suggested that PLZF might be one of the substrates of hPFTAIRE1 and/or
PCTAIRE1. All these studies shed light on the role of the two Cdc2-related
kinases, hPFTAIRE1 and PCTAIRE1, in the functional regulation of PLZF.

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