Cloning and Characterization of Casein Kinase 1

1
Cloning and Characterization of Casein Kinase 1
in Trichomonas vaginalis
Chia-hao Huang a and Petrus Tang a,b
a. Molecular Regulation and Bioinformation
Laboratory, Department of Parasitology, Chang
Gung University, Taoyuan 333, Taiwan. b. Chang
Gung Bioinformatics Center, Taoyuan 333, Taiwan.
Abstract Trichomonas vaginalis has an unique
cell cycle with a dominant G2 stage. The
regulation of cell cycle usually depends on the
activity kinases. We identified a T. vaginalis
expressed sequence tag (EST) clone (TvEST-14G2)
which showed sequence homology to casein kinase 1
(CK1). The 1572 bp complete open reading frame of
TvEST-14G2 encodes a protein of 524-amino acid
residues with predicted molecular weight of 63
kDa. Although the Trichomonad CK1 showed low
sequence identity at amino acid sequence level
with known CK1s, but simulated 3D model showed
high degree of similarity. Recombinant TvCK1 is
able to phosphorylate CK1-specific substrate. The
expression levels of TvCK1s in cold treated
synchronized cell cycle were followed by
real-time quantitative PCR. High level of TvCK1
expression was identified at G2 stage. TvCK1 may
play an important role in regulating the cell
cycle of T. vaginalis. Introduction
Trichomanas vaginalis, an anaerobic parasitic
protozoan, is the cause of trichomoniasis, the
second most sexually transmitted disease (STD)
(1). T. vaginalis represents one of the earliest
branching eukaryotic linages available for study
(2, 3, 4). In contrast with most eukaryotic
cells, the T. vaginalis G2 period was prolonged,
comprising 50 to 58 of the cell population (5).
Casein kinase 1 is a family of multipotential
Ser/Thr protein kinases common to all eukaryotic
cells. CK1 can phosphorylate a wide variety of
cellar proteins such as cytoskeleton components,
signaling molecules, metabolic enzymes, and
proteins involved in mRNA translation(6, 7). In
Plasmodium falciparum and Typansoma cruzi,
activity of CK1 is stage-specific regulated (8,
9). In Leishmania major CK1 regulates ectokinase
activity that capable of phosphorylating and
inactivating molecular of human complement system
(10). HRR25, CK1 of Schizosaccharomyce
cerevisiae, has been described involved in DNA
repair and cell cycle controll.
Fig. 5 Growth curves of T. vaginalis at different
temperatures. T. vaginalis with an initial cell
density of 2 x105 cells/ml was grown at 37oC (red
line) or 4oC (blue line). T. vaginalis was growth
arrested under cold treatment.
Fig. 8 Flow cytometery of T. vaginalis after cold
treatment. DNA content of T. vaginalis was
assessed using propidium iodide (PI) after cell
permeablilization with 1 Triton X-100 in PBS.
Cells were analyzed by FACScan and displayed
using WinMDI 2.9 software program. It showed that
cells undergo G1 phase at 1.5 hour.
Fig2. Comparison of 3D structure of Casein
kinases 1. Protein tertiary Structure modeling
was predicted by ExPASy Molecular Biology Server.
It shows that TvEST-14G2 had similar folding with
CK1s in human, mouse, yeast, Plasmodium
falciparum (PfCK1) and Trypanosoma cruzi (TcCK1.1
and TcCK1.2).Three casein kinase motifs are
showed in yellow.
A)
B)
M a b c d
Table 2. Expression of TvCK1 after recovering
growth. T. vaginalis total RNA were extracted
from cells recovered from cold treatment at 0,
0.5, 1, 1.5, 2, 2.5, 3 and 3.5 hour. Expression
levels of TvCK1 was determined by SYBR Green PCR
Core Reagents. TvEST-14G2 shows higher mRNA
expression at 1.5 hour (in red). TvCK1 was
expressed at the highest level at 1.5 hr, which
correspond to the appear of G1 cell population at
figure 8. TvCK1 may play an important role in
cell cycle regulation.
C)
a b c d 1 2 3
Fig 3. Overexpression of TvEST-14G2 fussion
protein. The TvEST-14G2 PCR product was cloned
into pet-15b vector. E. coli (BL21) extracts were
analyzed without (a) and with (b) 1mM IPTG
induction. After solubilization (supernatant
lane c, pellet d), the cell lysate was loaded
onto nikel-chelate
Fig. 6 Cell cycle stage of T. vaginalis under
cold treatment. T. vaginalis Culture grow at 4oC
were harvested each hour and analysed by flow
cytometery. DNA content of T. vaginalis was
determined by using propidium iodide (PI) after
cell permeablilization with 1 Triton X-100 in
PBS. Cells were analyzed by FACScan and displayed
using WinMDI 2.9 software program. T. vaginalis
under cold treatment showed G2 arrest after 4
hours.
Summary 1. The casein kinase 1 homologue,
TvEST-14G2, have been isolated from T.
vaginalis by EST and 5-RACE and encodes
524-amino acid residues with predicted
molecular weights of 63kDa. 2. Recombinant
TvEST-14G2 shows the activity that could
phosphorylate CK1-specific substrate. 3. Cell
cycle of T. vaginalis could be synchronized by
cold treatment and showed G2 period
arrest. 4. TvCK1 showed different expression
level in cell cycle and could be an
important factor in cell cycle regulating.
columns for affinity purification. The 6xHis
fusion protein were eluted (fraction 1 lane 1,
fraction 2 lane 2, fraction 3 lane 3). All
samples were analyzed in 8 SDS-PAGE by coomassie
blue (A and B) and anti-His antibody (C).

• Fig. 1 Isolation of full length TvEST-14G2.
  Strategy used to clone the full length
  TvEST14G2.(A) Product of 5 RACE of TvEST-14G2
  was checked on 1.5 agarose gel.(B) Total RNA was
  isolated from logarithmic phase of T. vaginalis
  cell culture was separated on 0.8 formamide gel
  (D) and transferred onto the Hybond-N membrane.
  The membrane was hybridized with the probe of
  TvEST-14G2 to analyze TvEST-14G2 transcripts
  length. (C)

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Table 1. Comparison of amino acid sequence
similarity. Similarity of amino acid sequence of
CK1s in various species are calculated by Vector
NT1 Suite. TvEST -14G2 showed low similarity
with other species.(in blue)
Fig. 4 Casein kinase activity of Recombinant
TvEST-14G2. 50 ng recombinant TVEST-14G2 (TvCK1)
or commercial rat casein kinase 1 react with or
without CK1 specific substrates at 30oC in 30
minis. 20µl reactions was dropped onto Whatman
P81 phosphocellulose paper, and washed by 75mM
otho-phosphoric acid. The counts are determined,
and the recombinant TvCK1 shows CK1 specific
activity. (in red)
Fig. 7 Recovering of Growth After Cold
Treatment. T. vaginalis is recovered at 37oC with
initial cell density 5 x 105 cells/ml after cold
treatment for 4 hours. The cells are harvested at
0.5 to 3.5 hour and analyzed by FACS.