Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform

Abstract

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon gamma, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

Figure 1. Colonic SPAK is a SPAK isoform that appears to be colon-specific

A) Amino acid sequence comparison of SPAK and colonic SPAK. Both SPAK and colonic SPAK isoform contain a potential caspase-3 cleavage site (DEMD) located between the kinase and regulatory domains, as well as a nuclear localization signal (RAKKVRR). Unlike other SPAK, colonic SPAK does not contain a PAPA box within its amino acid sequence. B) cDNAs obtained from Caco2-BBE cells, colon, liver, spleen, brain, prostate and kidney were subjected to PCR with primers specific to N-terminal part of SPAK. Caco2-BBE cells and colon tissues yielded a single ~330 bp product, while liver, spleen, brain, prostate and kidney samples yielded a single ~500 bp product.

Figure 2. Colonic SPAK is unusual splice single transcript isoform

A) Schematic representation of the SPAK gene intron-exon structure. The SPAK gene is composed of 18 exons spanning 184 kb long. In the exon 1 and 7, there are at the same time two unusual splice forms, in both the highly conserved GT nucleotides in the exon become the start of new and unusual intron, and they share the same end of intron nucleotides AG. The blue rectangle represents exons, the digits besides them are the size of it and order of the exon, and the triangles are introns with their respective number. The blue color sequence is the sequence in the exon, the black sequence is the predicted amino acid sequence. The outer black sequence is the related intron sequence start with GT and end with AG. B) Northern blot of the colonic SPAK. Lane 1 and lane 3, Caco2-BBE RNA were probed with probes exists in both colon and non-colon tissue. The lane 2 was probed with fragment of PAPA box which only in the non-colon tissue. The lane in the bottom represents the same loading control with 18 s RNA.

Figure 2. Colonic SPAK is unusual splice single transcript isoform

A) Schematic representation of the SPAK gene intron-exon structure. The SPAK gene is composed of 18 exons spanning 184 kb long. In the exon 1 and 7, there are at the same time two unusual splice forms, in both the highly conserved GT nucleotides in the exon become the start of new and unusual intron, and they share the same end of intron nucleotides AG. The blue rectangle represents exons, the digits besides them are the size of it and order of the exon, and the triangles are introns with their respective number. The blue color sequence is the sequence in the exon, the black sequence is the predicted amino acid sequence. The outer black sequence is the related intron sequence start with GT and end with AG. B) Northern blot of the colonic SPAK. Lane 1 and lane 3, Caco2-BBE RNA were probed with probes exists in both colon and non-colon tissue. The lane 2 was probed with fragment of PAPA box which only in the non-colon tissue. The lane in the bottom represents the same loading control with 18 s RNA.

Figure 3. Colonic SPAK isoform has serine/threonine kinase activity

A) In vitro transcribed/translated SPAK or kinase-deficient SPAK mutant proteins each had an apparent molecular weight of 53 kDa (lanes 1 and 2, respectively), while no protein was transcribed/translated from the Xpress vector alone (lane 4), lane 3 is the plasmid p38/pcDNA3 as control (43 kDa). B) Caco2-BBE cells expressing Xpress-tagged SPAK (lane 1), Xpress-tagged kinase-deficient SPAK mutant (lane 2) or Xpress vector alone were lysed, total proteins (40 μg/lane) were resolved by 4–20% SDS-PAGE, and blots were immunostained with an anti-Xpress antibody, which revealed an expressed protein with an apparent molecular weight of 60 kDa in Caco2-BBE cells expressing SPAK or kinase-deficient mutant SPAK-like. C) Anti-Xpress antibodies were used to immunoprecipitate in vitro transcribed/translated SPAK-like, the immunoprecipitates were subjected to 4–20% SDS-PAGE, and the blots were immunostained with mouse anti-phospho-ser (lane 1), anti phospho-thr (lane 2) or anti phospho-tyr (lane 3). Bands (~53 kDa) were detected with anti-phosphoser and anti-phospho-thr, but not anti-phospho-tyr, indicating that SPAK has the ability to autophosphorylate at ser and thr but not tyr. D) Protein kinase assays of immunoprecipitated in vitro transcribed/translated SPAK (lane 1), kinase-deficient SPAK (lane 2), or vector control (lane 3) were incubated with MBP for 30 minutes at room temperature and resolved by 4–20% SDS-PAGE. Blots were probed with anti-Xpress (Xpress-SPAK), anti-MBP (MBP) and anti-phosphorylated MBP (p-MBP).

Figure 4. Colonic SPAK isoform activates p38 MAP kinase signaling in intestinal epithelial cells

Total proteins from Caco2-BBE cells expressing Xpress-tagged SPAK (lane 1), Xpress-tagged kinase-deficient SPAK (lane 2), Xpress vector alone (lane 3) or relevant positive control treated cells (EGF for Erk, and Il-1 for JNK and p38) (lane 4) were lysed, and total proteins (40 μg/lane) were resolved by 4–20% SDS-PAGE and transferred to nitrocellulose membranes. The blots were immunostained with antibodies against Xpress (Xpress-SPAK), ERK (ERK), phospho-ERK (p-ERK), JNK (JNK), phospho-JNK (p-JNK), p38 (p38) and phospho-p38 (p-p38).

Figure 5. IFN-γ can increase SPAK mRNA expression in Caco2-BBE cells

Caco2-BBE cells were transfected with vectors encoding SPAK-like, and then treated with or without IFN-γ (1000 U/ml), TGF-β (10 ng/ml) or TNF-α (20 ng/ml) for 24 hours. For graphical representation of quantitative PCR data, the raw cycle threshold values (Ct values) obtained from the experimental groups were deducted from the Ct value obtained from the control group using the ΔΔCt method, and the data were normalized using 2^−ΔΔCt, with β-actin gene levels serving as the internal standard. Values are normalized as fold-differences relative to transcript levels in the untreated controls. The presented results are representative of three different experiments.

Figure 6. Cleaved N-terminal region of Colonic SPAK-isoform can translocate to the nucleus

Caco2-BBE cells were left untransfected (A), or were transfected with pcDNA3.1/V5 (vector tag alone; B), SPAK-pcDNA3.1/V5 (SPAK fused to a C-terminal V5 tag; C), SPAK-pcDNA6/Xpress (SPAK fused to an N-terminal Xpress tag; D) and kinase-deficient SPAK-pcDNA6/Xpress (kinase-deficient SPAK fused to an N-terminal Xpress tag; E), and then immunostained with anti-V5 (green), anti-Xpress (green), or rhodamine phalloidin (actin, red). The presented images represent horizontal sections (xy) taken from below the apical plasma membrane of transfected polarized Caco2-BBE monolayers.

Figure 7. Colonic SPAK-isoform over-expression decreases the barrier function in intestinal epithelium

TER was measured in Caco2-BBE cells transfected with colonic SPAK-isoform-pcDNA6/Xpress (colonic SPAK isoform), pcDNA6/Xpress vector alone (vector) and kinase-deficient SPAK-like-pcDNA6/Xpress (kinase-deficient colonic SPAK isoform). The trace represents TER values collected at 5s intervals for 10 minutes after a 20 minute equilibrium period, and is representative of six experiments.