Identifying and characterizing a novel protein kinase STK35L1 and deciphering its orthologs and close-homologs in vertebrates

Abstract

Background: The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria.

Methodology/principal findings: In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes.

Conclusions/significance: We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.

Figure 1. EGFP-STK35 (Clik1) does not translocate to actin stress fibers upon mRFP-CLP36 coexpression.

Endothelial cells were transfected with EGFP-STK35 plasmid alone (A) or cotransfected with mRFP-CLP36 (B). The nuclear localization of EGFP-STK35 protein (A, green) was not affected by coexpression of mRFP-CLP36 (B1). mRFP-CLP36 (Red) was mainly localized on actin stress fibers (B2).

Figure 2. Expression analysis of STK35.

(A) To verify the expression of STK35 mRNA, RNA and cDNA were prepared from endothelial, HEK293 and HeLa cells. 273 bp PCR products were amplified with STK35 specific primers from cDNA pools of endothelial, HEK and HeLa cells. β-actin amplification was used as a control. The primer pairs correspond to different exons to ensure the amplification from mature RNA. (B) Endothelial cells (EC) were transiently transfected with plasmids encoding different constructs of EGFP-STK35. Cell lysates of transfected or untransfected EC were immunoblotted with goat anti-STK35 antibody, or with mouse anti-EGFP as expression control. The detected bands correspond to the size of the fusion proteins (EGFP-STK35_Δ72-400: 35 kDa; EGFP-STK35_Δ1-6: 66 kDa; EGFP-STK35:72 kDa. The construct lacking the C-terminal part (EGFP-STK35_Δ72-400) was not recognized by anti.STK35 antibody. Note the additional band at 58 kDa in all lanes blotted with the anti-STK35-antibody (arrow). (C) Nuclei of EC, HEK and HeLa cells were isolated, and lysates were immunoblotted with goat anti-STK35 antibody or goat IgG as control. The bands detected by the antibody could not be found with the goat IgG control. In EC, HEK and HeLa nuclear lysates and in the whole cell lysate of EGFP-STK35 transfected cells a band of 58 kDa could be detected. In EGFP-STK35 (72 kDa) transfected cells, additional band of 72 kDa was detected. EC were transfected with pooled STK35L1 siRNA or control siRNA. After 24 to 72 hours of transfection, cell lysates were examined by western blotting with anti-STK35 antibody. After 72 hours, expression of 58 kDa band of STK35 protein was reduced by 80%. Actin blot was performed as control for equal loading.

Figure 3. Genomic analysis of STK35L1.

(A) Schematic representation of the published STK35 (Clik1) gene. The location on chromosome 20 is shown. The coding region is shown in blue. (B) The structure of the STK35 locus after identification of the putative TSR. The 1035 bp sequence between TSR and the start codon of the published gene is highly conserved among mammals as shown in % identity. (C) Schematic representation of the STK35L1gene. The 5′ extended sequence is marked in light blue. (D) The putative start codon of the STK35L1 gene matches the Kozak consensus sequence RccAUGG. Some nucleotides in this sequence are more important than others. For a ‘strong’ consensus, the nucleotides at positions +3 (G) and −3 (R = A or G) must both match the consensus. . E) mRNA expression of the STK35L1 gene. Upper panel: Amplification of the expressed transcripts of STK35L1 from human EC cDNA. The lower axis: the putative mRNA transcript of STK35L1 gene and its length in base pairs. The extended sequence to the 5′ end is shown in light blue, the previously published sequence is represented in dark blue. Positions of introns are indicated as white bars. Arrows over the axis represent the relative position of primers used for the amplification of cDNA. The sizes of the three obtained PCR products are indicated.

Figure 4. Alignment of protein sequence of STK35L1 from different vertebrates human (HSAP), mouse (MMUS) rat (RNOR), and zebrafish (DRER).

Red arrow indicates the newly identified start codon while blue arrow head indicates start codon previously published. The position of kinase domain is indicated by blue line. The STK35L1 kinase domain of zebrafish has 81% identity with human kinase domain.

Figure 5. Knockdown of STK35L1 protein expression.

To knockdown the STK35L1 protein expression, EC were transfected with pooled STK35L1 siRNA or control siRNA. After 24 to 72 hours of transfection, cell lysates were examined by western blotting with anti-STK35 antibody. After 72 hours, expression of 58 kDa band of STK35 protein was reduced by 80%. Actin blot was performed as control for equal loading.

Figure 6. Subcellular distribution of STK35L1 and its mutants in endothelial cells.

Cells transfected with EGFP-STK35L1 or deletion mutants were fixed and DNA was stained with Hoechst dye. EGFP-NSTK35L1 is mainly localized in nucleus and nucleolus (left panel white arrow). The nucleolus is excluded by Hoechst-dye staining (middle panel white arrow). In contrast, EGFP-STK35 is mainly localized in nucleus. EGFP-Kinase (N-terminal 1–196 amino acids of STK35L1 were deleted) is diffusely distributed in the nucleus and the cytoplasm indicating that N-terminal part of STK35L1 has functional nucleolar localization signal.

Figure 7. Genomic localizations of STK35L1 and flanking genes in different vertebrates.

Figure 8. Genomic localizations of STK35L2 and flanking genes in different vertebrates.

Figure 9. Genomic localizations of STK35L3 and flanking genes in different vertebrates.

Figure 10. Evolutionary relationships of STK35L1 and its homologs with selected kinases.

The previously published probable STK35 gene of Sea urchin is not STK35 gene as do not cluster with STK35 like genes from other genomes with 60% bootstrap values. In contrast, Ciona STK35 gene is clustered with STK35 like genes. The evolutionary history of selected kinases was inferred using the Neighbor-Joining method . The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of these selected kinases . Branches corresponding to partitions reproduced in less than 60% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated kinases clustered together in the bootstrap test (1,000 replicates) are shown above the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 227 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4. Outgroup is yeast STE11 gene choose from www.kinase.com. SCER–yeast, HSAP–human, DRER- zebrafish, MDOM–opossum, CINT–sea squirt, SPUR–sea urchin.

Figure 11. Evolution of STK35L1 homologs in different metazoan genomes.

These genes most probably originated from a STK35L1 homolog from sea squirt. This finding dates the origin of STK35L1 homologs to 550 MY ago. The STK33 gene (28% identity with STK35L1 in the kinase domain) is found already in the sea anemone genome (accession id e_gw.81.82.1), dating ∼700 MY ago. The estimated divergence times (in MY) were taken from and are marked with red arrows. +, presence, -, absence, *, ancestor gene. ?, information not available.

Figure 12. Variable exon-intron organization of STK35L1 homologs and their ancestor gene from Ciona.

Exons are depicted by boxes and introns by broken lines and labeled with their indicated sizes (bp). An extension of 603 bp of STK35 gene comprising of full exon 1 and part of exon 2 codes the N-terminal extension of the protein (marked in grey color). Kinase domains are indicated by black line.