- Research article
- Open Access
Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum
- Shide Liu†1,
- Zhuolong Zhou†1,
- Ziyang Lin2,
- Qiuling Ouyang1,
- Jianhua Zhang1,
- Shengli Tian1 and
- Miao Xing1Email author
© Liu et al; licensee BioMed Central Ltd. 2009
- Received: 28 February 2009
- Accepted: 25 August 2009
- Published: 25 August 2009
Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study.
We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus.
This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.
- Space Sequence
- L929 Cell
- Nuclear Localization Signal
- Cyan Fluorescent Protein
- Physarum Polycephalum
Serine/arginine (SR) protein-specific kinases (SRPKs) represent a class of evolutionarily conserved kinases that specifically phosphorylate the arginine/serine-rich (RS) domains of the SR splicing factor . After the identification of SRPK1–the first SRPK–by Gui et al. [2, 3], other SRPKs such as SRPK2 , mouse SRPK1 and SRPK2 , yeast Dsk1  and Sky1p , nematode SPK-1 , Trypanosoma cruzi TcSRPK , and Arabidopsis thaliana SRPK4  have been subsequently identified. Some studies have shown that SRPKs are mainly localized in the cytoplasm, with only a few present in the nucleus [[2, 5], and ]. The SRPKs that are localized in the cytoplasm regulate the nuclear import of SR proteins via phosphorylation , while those in the nucleus regulate the nuclear localization of the SR splicing proteins via phosphorylation [13–15]. Ding et al.  have discovered that the spacer sequences present between the conserved domains of mammalian SRPKs have cytoplasmic anchoring function. However, there has been no report on the nuclear localization signal (NLS) of SRPKs.
Model organism Physarum polycephalum is mitochondria-containing primitive eukaryotes. Its life cycle includes a single-celled amoeba, plasmodium (the main life form), and sporulation stages. The nuclei in the same plasmodium proliferate by way of synchronization of mitosis. In our previous study, we identified an SRPK containing 426 amino acids (aa) from P. polycephalum; this kinase was termed PSRPK (GenBank accession No. DQ140379). Similar to other SRPKs, PSRPK also has 2 conserved domains and can phosphorylate human SR protein alternate splicing factor/splicing factor 2 (ASF/SF2). However, it differs from other SRPKs in that the divergent motif (≥ N) in its N-terminus is rich in acidic amino acids; the spacer sequence of PSRPK between two conserved domains is shorter than that of other SRPKs. In this study, the distribution of PSRPK fluorescent fusion protein in mammalian cells was observed using laser scanning confocal microscopy. Maximum fluorescence was detected in the nucleus, suggesting the existence of an NLS in PSRPK. When the distribution of truncated PSPRK peptides was observed, a putative NLS was found in the C-terminal of PSRPK based on its homology to the classic NLS. When the sequence was deleted or when lysine 320 in the sequence was substitute by threonine, the cytoplasmic distribution of PSRPK was observed.
Construction of expression plasmids containing PSRPK and truncated PSPRK peptides
Primers used for cloning psrpk and tps
sense and anti-sense primers
R1: 5'-ACGCGTCGAC AGAAATGGAGGCACATCAGCC-3'
R3: 5'-ATGCGGATCC GCCAGAAATGGAGGCACATCAGCC-3'
F4: 5'-CCGGAATTC ATGGAAAACATATTCAAGGAGAAG-3'/R2
Construction of expression plasmids containing mutant and default PSRPK peptides
Primers used for cloning psrpk d and psrpk m
Sense and anti-sense primers
959A → 959C
5'-GGAGATCTCCTTTTTTGC CCCAAAAC AGGAG-3'/R3
Transfection of mammalian cells with lipofectamine
The abovementioned recombinant plasmids were used to transform E. coli DH5a cells. The positive recombinant products were grown in Luria-Bertani (LB) medium containing 30 μg/ml kanamycin. The plasmids were isolated using the alkaline lysis method, dissolved in Tris-EDTA (TE) buffer, and quantified using GeneQuant pro (Amersham Bioscience, GE Healthcare). The purified plasmid (A260/A280 > 1.8) was diluted to 16 μg/ml in serum-free RPMI-1640 medium (Gibco). Lipofectamine™ 2000 (Invitrogen) was diluted in serum-free RPMI-1640 medium (40 μl/ml) and then mixed with an identical volume of plasmid solution, resulting in lipid-DNA complexes.
HeLa and L929 cells (ATCC) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator. The cells were harvested while they were in the logarithmic phase and 1.5 ml culture (approximately 1 × 105 cells) was seeded into a 35-mm plate with a coverslip and cultured for 24 h. The medium was replaced with serum-free RPMI-1640 medium, and the cells were cultured for 1 h to initiate transfection. The cells were overlaid with 200 μl lipid-DNA complexes and cultured for 5 h. The medium was replaced with 1.5 ml of 10% FBS medium, and the cells were further cultured for 48 h.
Staining of cells with 4',6-diamidino-2-phenylindole (DAPI)
The HeLa cells were transfected with pECFP-psrpk, and L929 cells were transfected with pECFP-psrpk, pDsRed-psrpk, pDsRed-tp5~pDsRed-tp5, pDsRed-psrpk d and pDsRed-psrpk m as described above, respectively. The transfected cells were incubated on a coverslip for 48 h, following which DAPI staining (Bitian) was performed for 5 min. The cells were washed 3 times (each wash, 5 min) with D-Hands solution to remove DAPI. Fluorescence was visualized under a fluorescence microscope (Olympus BX51, 400×) or a confocal microscope (Leica TCS SP2, 1000×) at 465~495 nm (green) or 560~600 nm (red) in order to observe the distribution of the fluorescent fusion protein in the cells.
Three-Dimensional Structure Modeling of PSRPK
The three-dimensional (3-D) PSRPK structure was modeled by the Swiss Institute of Bioinformatics program SWISS-MODEL, available at http://swissmodel.expasy.org/, and the results analyzed by the DeepView program .
PSRPK was mainly localized in the nucleus of mammalian cells
Existence of an NLS on the C-terminal conserved domain of PSRPK
PSRPK NLS was located in the 318PKKGDKYDKTD328 sequence
The NLS sequence of PSRPK contained a Ω-loop motif
PSRPK has an NLS sequence
Ding et al.  studied the cell localization of mammalian SRPKs and found that almost all SRPKs without spacer sequences are accumulated in the nucleus, indicating that there is a cytoplasm localization signal in the spacer sequence between the conserved domains. Kuroyanagi et al.  speculated that mouse SRPK1 might have 2 potential NLSs that are located in 11~21 aa and 265~277 aa; thus, mSRPK2 may have a potential NLS in 264~276 aa. However, direct evidence of NLS sequences in SRPK family members has been lacking thus far. Laser scanning confocal microscopy revealed that RFPs of default PSRPKs containing 318PKKGDKYDKTD328 mainly accumulated in the nucleus of mammalian cells, while the PSRPKs in which the abovementioned sequence was deleted did not accumulate in the nucleus. This indicated that the NLS of PSRPK was located in the 318PKKGDKYDKTD328 sequence of the C-terminal conserved domain.
Further, Ding et al.  showed that mammalian SRPKs were mainly distributed in the cytoplasm. However, the results obtained in our study showed that PSRPK was mainly expressed in the nucleus of the mammalian cells. A comparison of the primary structure of PSRPK and other SRPKs revealed that the conserved domains were almost identical; however, the nonconserved ≥ N and spacer sequence differed among the kinases. A cytoplasm localization signal was present in the spacer sequences of mammalian SRPKs. Further, the spacer sequence of PSRPK was considerably smaller than that of mammalian SRPKs. This difference might be the major reason why PSRPK cannot anchor itself in the mammalian cytoplasm.
Function of the PSRPK NLS is related to the loop motif
A classic NLS (cNLS) comprises a monopartite or bipartite signal. A monopartite NLS contains 1 cluster of basic residues, while a bipartite NLS contains 2 clusters of basic residues . Similar to the sequence feature of a monopartite NLS, the NLS of PSRPK is rich in basic residues. A monopartite NLS sequence that is located at the terminal of a protein is usually in a coil, while that located in the interior of a protein is usually in a loop. The PSRPK NLS is also located in the basic loop between 2 α-helixes (Figure 5). The close side chains of K320 and D325 form a salt bridge, thus forming a stable NLS Ω-loop motif (Figure 5C). The experimental results showed that PSRPK lost its nuclear localization ability following the deletion of 318 PKKGDK323 or mutation from K320 to T320. The deletion or mutation destroyed the Ω-loop motif of PSRPK, thus suggesting that the loop structure of PSRPK NLS controls the nuclear localization of PSRPK. The 318PKKGDKYDKTD328 sequence, which corresponds to the nonconserved sequences of SRPK1 and Sky1p, is located in the loop of an HLHM [18, 19] (Figure 5B). Therefore, studies on NLSs of SRPKs should focus on the loops in HLHMs.
In this study, by truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.
The work was supported by the National Natural Science Foundation of China (30470113), Natural Science Foundation of Guangdong Province (04011314)
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