Copyright ©The Histochemical Society, Inc.

Profiles of PrKX Expression in Developmental Mouse Embryo and Human Tissues

Wei Li, Zu-Xi Yu and Robert M. Kotin

Laboratory of Biochemical Genetics (WL,RMK) and Pathology Core (Z-XY), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

Correspondence to: Robert Kotin, Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg. 10, Rm. 7D05, 10 Center Drive, Bethesda, MD 20892. E-mail: kotinr{at}nhlbi.nih.gov


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Protein kinase X (PrKX), karyotypically located on the human X chromosome, is a type I cAMP-dependent protein kinase. Although a specific role for PrKX has not yet been defined, PrKX gene expression in mouse and human tissues has been profiled only by in situ hybridization and Northern blot analyses and not by protein expression. To determine more precisely the PrKX protein levels, we developed specific anti-PrKX antibodies and examined gestationally staged mouse embryo sections by immunohistochemistry. These results showed that PrKX is ubiquitously distributed and highly expressed in murine central nervous system and heart tissues in early developmental stages and in most organs at later stages but was not detected in either connective tissues or bone. Using Western blots to detect PrKX, total protein extracts from eight different adult or fetal human tissues including brain, heart, kidney, liver, lung, pancreas, spleen, and thymus were analyzed. Although PrKX protein was present in each of the tissues tested, the protein levels varied depending on tissue type and developmental stage. Very low protein levels were found in heart tissues from a 5-month-old fetus and from an adult, whereas PrKX proteins were more abundant in fetal brain, kidney, and liver tissues compared with adult samples of the same tissue type. (J Histochem Cytochem 53:1003–1009, 2005)

Key Words: PrKX • type I cAMP-dependent protein • kinase • immunohistological staining • Western blot


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
ADENOSINE 3',5'-cyclic monophosphate (or cyclic adenosine monophosphate or cAMP) is implicated in a number of cellular processes including transduction of extracellular signals and maintenance of cellular homeostasis. Most of the known effects of cAMP in eukaryotic cells are mediated by cAMP-dependent protein kinases. The inactive holoenzyme of protein kinase A (PKA) consists of two regulatory (R) subunits and two catalytic (C) subunits. Four R-subunit isoforms and three C-subunit isoforms have been identified in human cells (Skalhegg and Tasken 2000Go), but only two C-subunit isoforms have been isolated from mouse cells (McKnight 1991Go). Some isoforms appear to have tissue-specific expression patterns (C{gamma}, RI{gamma}, RII{gamma}) (Scott 1991Go), whereas other isoforms appear ubiquitously expressed (e.g., C{alpha}, Cß, RI{alpha}, RII{alpha}) (Beebe et al. 1990Go; Scott 1991Go). Binding of cAMP to the two sites on each of the R-subunits causes the release of active C-subunits that can then phosphorylate serine and threonine residues on substrate proteins.

Recently, the cDNAs encoding the novel X-chromosome-encoded cAMP-dependent protein kinase, PrKX, was cloned from both human (Klink et al. 1995Go) and mouse (Blaschke et al. 2000Go). Characterization of PrKX demonstrated that it is a type I R-subunit regulated kinase (Zimmermann et al. 1999Go; Li et al. 2002Go) with a much lower catalytic activity toward either a synthetic substrate peptide of PKA referred to as kemptide or histone H1 protein, a physiological substrate of PKA (Di Pasquale and Stacey 1998Go; Zimmermann et al. 1999Go). Overall, at the protein level, the catalytic subunit of PrKX has 50.2%, 50.8%, and 44.83% identity with the catalytic subunit, C-subunit of PKA{alpha}, PKAß, and PKA{gamma}, respectively. Not surprisingly, within the core catalytic region, PrKX and PKA are highly conserved, whereas the N-terminal and C-terminal regions retain between 21.7 and 30.9% identity, depending on the PKA isoform (Zimmermann et al. 1999Go; Li et al. 2002Go).

An interesting association exists between PrKX and a human dependovirus, adeno-associated virus (AAV) non-structural proteins. The unspliced non-structural proteins, Rep78 and Rep 52, bind to the catalytic subunits of PKA and PrKX, apparently acting as a pseudosubstrate, thus inhibiting kinase activities (Chiorini et al. 1998Go; Schmidt et al. 2002Go; Di Pasquale and Chiorini 2003Go). An explanation for these interactions is not readily apparent. Although transient expression assays have shown that PrKX is capable of activating CREB-dependent transcription similar to PKA (Di Pasquale and Stacey 1998Go; Li et al. 2002Go), functional differences have also been reported (Semizarov et al. 1998Go; Li et al. 2002Go). Renal expression of PrKX is developmentally regulated (Li et al. 2002Go). Transiently overexpressing PrKX in vitro activates migration of FIB4 cells, a PKA-deficient porcine renal epithelial cell line, and also causes branching morphogenesis of MDCK cells (Li et al. 2002Go). Interestingly, none of these effects was observed for PKA (Li et al. 2002Go). PrKX gene expression is upregulated in HL-60 cells that have been stimulated to differentiate into granulocyte, monocyte, and macrophage lineages (Semizarov et al. 1998Go; Junttila et al. 2003Go). PrKX gene expression also appears critical for monocyte and macrophage maturation as well as human myeloid leukemia HL-60 and mouse myeloid follicular dendritic cell lines' differentiation (Semizarov et al. 1998Go; Junttila et al. 2003Go).

Subcellular compartmentalization provides another level of regulation affecting the activation of the kinases by cAMP and access to substrates (Barradeau et al. 2002Go). Type I kinases are mainly cytoplasmic, whereas type II kinases are typically found associated with membranes and subcellular organelles (Rubin 1994Go). The expression patterns for the R- and C-subunits of PKA have been characterized at the level of transcripts in the central nervous system (CNS) of 14-day-old mouse embryos using in situ hybridization (Cadd and McKnight 1989Go). PrKX mRNA expression during mouse embryonic development and tissue distribution in adults have been analyzed using the same strategy (Blaschke et al. 2000Go). Protein expression profiles of either PKA or PrKX during mouse embryogenesis have not been described. Although PrKX mRNA expression has been characterized in previous studies, mRNA levels indirectly represent the content of translated protein product, whereas immunohistochemical and Western blot detection provide more accurate measurements of temporal expression and relative quantitative levels of protein antigens.

In this study, we examined the distribution of PrKX during mouse embryonic development using a specific antibody developed in our lab. In addition, the expression of PrKX protein in human adult and fetal tissues was also analyzed by Western blot.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Production of PrKX Antisera
Peptides corresponding to 21 amino acid C-terminal fragments of mouse PrKX (residues 335–355) or human PrKX (residues 338–358) were synthesized, purified to 98% homogeneity, and then conjugated to keyhole limpet hemocyanin (KLH) through a cysteine residue added to the N terminus during peptide synthesis. New Zealand white rabbits were immunized intraperitoneally with 200 µg of KLH–peptide conjugate in Freund's complete adjuvant and boosted three times intradermally with 100 µg of antigen biweekly before the first bleeding and subsequently every week with the same amount of antigens before the second and third bleeding. Antisera were aliquoted and stored at –20C. Antiserum production services were provided by Sigma Genosys Ltd. (Woodlands, TX).

Characterization of the Specificity of PrKX Antibodies
PrKX and PKA were obtained from two sources for characterizing the PrKX polyclonal antibodies. PrKX and PKAC{alpha} were expressed as 6x His fusion proteins in African green monkey COS1 cell line and purified with Ni-NTA resin (Novagen Corp.; Madison, WI) (Zimmermann et al. 1999Go). GST-PrKX and GST-PKAC{alpha} fusion proteins were expressed in yeast and purified with glutathione beads (Zimmermann et al. 1999Go). The GST fusion proteins were fractionated by polyacrylamide gel electrophoresis (PAGE) with 4–12% Bis-Tris SDS-PAGE gels (Invitrogen Life Technologies; Carlsbad, CA) and electrotransferred onto 0.2-µm pore-size polyvinylidene fluoride (PVDF) membranes (Invitrogen Life Technologies). The membranes were preincubated in PBST (phosphate-buffered saline containing 0.1% Tween-20) with 4% non-fat dry milk, then incubated overnight at 4C with PrKX antisera or preimmune sera diluted 1:400 in PBST with 4% non-fat dry milk. The membranes were washed three times (10 min each time) with PBST and then incubated with a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (Amersham Biosciences; Piscataway, NJ) diluted 1:5000 in PBST with 4% non-fat dry milk. The membranes were washed as above to remove unbound secondary antibody. The antibody complex was detected with HRP using chemiluminesence substrates (ECL reagents; Amersham Biosciences). Where indicated, the blots were stripped by incubating the membrane in stripping solution (2% w/v SDS, 100 mM 2-mercaptoethanol, 62.5 mM Tris-Cl, pH 6.7) at 50C for 30 min and reprobed using GST (1:10,000) and His antibodies (1:250) or PKA antibody (1:250). Antibodies for PKA (Sc-903), GST (Sc-459), and His-tag (Sc-804) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The specificity of the human and mouse PrKX antiserum was also examined using human Hela cell lysates and mouse NIH3T3 cell lysates.

Immunohistochemical Analysis of Mouse Embryo Sections
Tissue sections of stage 9.5- to 18.5-day-old mouse embryos were commercially obtained (Paragon Bioservices; Baltimore, MD). Briefly, the tissue was fixed with 10% buffered formalin and embedded in paraffin. Five-µm-thick sagittal sections were cut and mounted on silane-coated glass slides. For the immunostaining, indirect peroxidase method was applied. After routine deparaffination and rehydration through gradient ethanol immersions, the slides were then steam heated for 20 min to expose the antigen. Endogenous peroxidase activity was quenched by using 3% (v/v) H2O2 followed by three 5-min washes in PBS containing 0.2% (v/v) Triton X-100, and the sections were blocked with 10% (v/v) normal goat serum in PBS. Specimens were incubated for 1 hr with rabbit anti-PrKX serum (1:500) diluted in PBS containing 0.3% (v/v) Triton X-100 and 0.1 BSA (w/v) followed by three 5-min washes in PBS before incubation for 30-min with HRP-conjugated goat anti-rabbit F(ab')2 fragments (KPL; Gaithersburg, MD). Negative control was performed with omitted primary antibody. The samples were washed as above and the immunoreactivity was visualized with DAB (3,3'-diaminobenzidine) substrate kit (Vector Laboratories; Burlingame, CA). Specimens were counterstained with hematoxylin for 30 sec and washed with tap water. The sections were immediately dehydrated by sequential immersion in gradient ethanol and xylene and then mounted with Permount and coverslips. Images were obtained from Leica DMRAX upright microscope coupled with a digital camera (Leica; Bannockburn, IL).

Northern Blot Analysis of PrKX Gene Expression in Human Fetal Tissues
The distribution of PrKX mRNA in human tissue was determined by Northern blot using a preblotted MessageMap membrane (Stratagene; La Jolla, CA) consisting of twice selected poly(A)-containing RNA (2 µg/lane) from five human fetal tissues (brain, heart, kidney, liver, and lung). The PrKX cDNA containing the open reading frame was PCR amplified from a plasmid DNA (I.M.A.G.E. Clone ID 3596013; ATCC, Manassas, VA) and agarose-gel purified. Uniformly radiolabeled probe was produced by random priming with [32P]dCTP to a specific activity of 1.8 x 109 dpm/µg (Rediprime II Random Prime Labeling System; Amersham Biosciences). The labeled probe (25 ng) was denatured by boiling for 2 min and used for the hybridization in a total volume of 5 ml hybridization solution according to manufacturer's directions (MiracleHyb; Stratagene). The blots were hybridized with probe at 68C overnight. Unhybridized probe was removed by washing twice for 15 min each at room temperature with 2x SSC solution containing 0.1% SDS followed by a high stringency wash with 0.1x SSC solution and 0.1% SDS for 30 min at 60C. The membrane was covered with plastic film and exposed overnight to X-ray film (XAR; Kodak, Rochester, NY) with intensifying screen at –80C. For rehybridization, the membrane was stripped of probe by washing with boiling washing solution (0.1x SSC and 0.1% SDS) twice for 15 min each and hybridized with 10 ng of a ß-actin probe labeled as above to standardize mRNA loading between samples and for assessing the PrKX mRNA level.

Western Blot Analysis of PrKX Expression in Human Tissues
Proteins isolated from either adult or fetal tissues including brain, heart, kidney, liver, lung, pancreas, spleen, and thymus were commercially obtained (Biochain Institute; Hayward, CA). According to the manufacturer, the protein concentrations from each tissue were determined using BCA protein assay. To prepare the Western blot, 8 µg of each sample was mixed with 2x SDS sample buffer containing 5% 2-mercaptoethanol, boiled for 5 min, cooled to room temperature, and loaded onto 4–12% Bis-Tris SDS-PAGE gels. Following electrophoresis (180 V, 50 min), the gels were electroblotted onto PVDF membranes. Primary anti-human PrKX antiserum was diluted 1:400 into PBST containing 4% non-fat milk.


    Results
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 Materials and Methods
 Results
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 Literature Cited
 
Genomic sequence analysis revealed that PKA catalytic subunits are the most homologous cellular proteins to PrKX. The specificity of the antiserum raised against C-terminal epitope of either mouse or human PrKX was tested by immunoblotting using purified PrKX and PKA fusion proteins as antigens. Both mouse and human PrKX antibodies recognized PrKX protein at 1:400 dilutions but failed to cross-react with PKA (Figure 1A) . PKA antibody against the C-terminal 20 aa epitope also did not cross-react with PrKX protein (Figure 1B). Comparable amounts of recombinant PrKX and PKA proteins, by mass, were loaded onto the membranes as shown by Western blots with both anti-6x His and anti-GST antibodies (Figure 1C). As a negative control, membranes blotted with PrKX and PKA proteins detected with preimmune sera did not yield positive signals (data not shown). Also, no cross-reactivity with other cellular proteins in Hela cells and NIH3T3 cells was found (data not shown). Therefore, this antiserum was subsequently used as a reagent to detect PrKX either by in situ immunostaining or Western blotting.



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Figure 1

Characterization of rabbit antiserum against mouse and human protein kinase X (PrKX). Purified proteins, either His or GST fusion proteins, were transferred to membranes and incubated with the indicated antiserum. Approximately 500 ng of each protein per lane was applied for electrophoresis. The proteins were loaded in the same order for each panel. Lane 1: His-tagged mPrKX. Lane 2: GST-mPrKX. Lane 3: His-tagged mPKA. Lane 4: GST-mPKA. Lane 5: His-tagged hPrKX. Lane 6: GST-hPrKX. Lane 7: His-tagged hPKA. Lane 8: GST-hPKA. (A) Western blots incubated with anti-PrKX anti-sera. Lanes 1–4: membrane incubated with anti-mouse PrKX antibody; Lanes 5–8: membrane incubated with anti-human PrKX antibody. (B) Western blots incubated with anti-PKA antibody. Lanes 1–4: membrane incubated with anti-mouse PKA antibody; Lanes 5–8: membrane incubated with anti-human PKA antibody. (C) Western blots incubated with both anti-His- and anti-GST-specific antibodies.

 
To examine the patterns of PrKX expression during mouse embryo development, sagittal sections of mouse embryos between days 9.5 and 18.5 postcoitus (pc) were immunostained. PrKX expression appears ubiquitous from day 9.5 pc using the rabbit anti-mouse PrKX antiserum with noticeably higher levels found in the brainstem, vascular endothelial cells, and heart as represented by the 12.5-day mouse embryo sections (Figure 2A) . The negative control showed no positive reaction (Figure 2B).



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Figure 2

Immunohistochemical staining of 12.5-day mouse embryo sagittal section. Mouse embryo sections were incubated with either antiserum against mouse PrKX at 1:500 dilutions (A) or with normal rabbit serum diluted 1:500 as a negative control (B). Immunoreactivity was found in the brain and heart with anti-PrKX antibody, but there was no reaction in the negative control. Bar = 1 mm.

 
More detailed examination shows that PrKX appears at day 9.5 in the mouse brain and heart, increasing by day 12.5, and then remaining at this level until day 18.5 (Figure 3) . PrKX expression was also apparent in lung, liver, thymus, pancreas, kidney, and adrenal gland as well as in skeletal muscle after 14.5 days (Figure 4) . However, there was no staining found in connective tissues, particularly cardiac valves, intestine and kidney, and bones. Although the experiments were not designed for quantitative analysis, the relative levels of PrKX are apparent.



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Figure 3

Immunohistochemistry showing the PrKX expression in developmental mouse. (A) Detectable PrKX in the heart and brain at day 12.5. (B) Immunoreactivity for PrKX increased in 16.5-day embryos. Bars = 1 mm.

 


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Figure 4

Expression of PrKX in organs of mouse embryos (14.5 days in A–C and 16.5 days in D). (A) Brain; (B) heart, thymus, and lung; (C) liver and pancreas; and (D) kidney, adrenal gland, and skeletal muscle. Bar = 0.5 mm.

 
Previous publications have presented conflicting data on PrKX gene expression in different human tissues (Klink et al. 1995Go; Semizarov et al. 1998Go). To characterize the mRNA level in major human fetal organs, we performed Northern blot analysis using poly(A)-selected mRNAs extracted from fetal brain, heart, kidney, liver, and lung. Two transcripts were detected, a major band corresponding to ~6.1 kb and a minor band of ~2.4 kb (Figure 5) . The sizes of these bands are consistent with previously reported transcripts (Genebank accession NM_005044 and BC041073). The transcripts were detected in each of the five tissues analyzed with the most abundant mRNA level observed in fetal brain. Intermediate levels were detected in kidney, lung, and heart, whereas the lowest mRNA level was found in liver. With the exception of heart mRNA, our results are in agreement with those published by others (Klink et al. 1995Go).



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Figure 5

Northern blot analysis of PrKX mRNA expression in fetal human tissues. A Northern blot of five human fetal tissues (Stratagene; 2 µg poly(A)-containing RNA per lane) was hybridized with 32P-labeled PrKX probe (1 kb). A major transcript ~6.1 kb and a minor transcript ~2.4 kb were detected. Lane 1: Brain. Lane 2: Heart. Lane 3: Kidney. Lane 4: Liver. Lane 5: Lung.

 
By Western blot analysis, we compared the relative levels of PrKX protein in the following eight different fetal and adult human tissues: brain, heart, kidney, liver, lung, pancreas, spleen, and thymus. The Western blot (Figure 6) shows abundant PrKX proteins in all fetal tissues represented, except the 20-week-old fetal heart. Among adult tissues characterized by Western blot, the PrKX protein concentrations were below the detection limit both in heart and liver tissues, demonstrating that the PrKX levels in fetal brain, kidney, and liver were significantly higher in fetal tissues than in adult tissues.



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Figure 6

Western blot analysis of PrKX in fetal human tissues. Total protein derived from eight human fetal and adult tissues (8 µg total proteins per lane) was immunoblotted using a specific antibody against human PrKX protein. A band corresponding to ~41 kDa was observed. Lane 1: Fetal liver. Lane 2: Adult liver. Lane 3: Fetal kidney. Lane 4: Adult kidney. Lane 5: Fetal heart. Lane 6: Adult heart. Lane 7: Fetal brain. Lane 8: Adult brain. Lane 9: Fetal thymus. Lane 10: Adult thymus. Lane 11: Fetal spleen. Lane 12: Adult spleen. Lane 13: Fetal pancreas. Lane 14: Adult pancreas. Lane 15: Fetal lung. Lane 16: Adult lung.

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
PrKX is a type I cAMP-dependent protein kinase and, although the kinase shares some similar characteristics with PKA, functional differences have been established (Semizarov et al. 1998Go; Li et al. 2002Go; Junttila et al. 2003Go). Immunohistochemical staining of staged mouse embryos demonstrated ubiquitous expression patterns of PrKX with notably high levels of protein in the developing heart and CNS system. The increased mRNA level of PrKX in differentiating neurons has previously been observed by in situ hybridization (Blaschke et al. 2000Go). Therefore, it is likely that PrKX plays some role in mouse heart and CNS development. It has been reported that type I regulatory subunit (RI) knockout mice were unable to develop functional heart tube at day 8.5 (Amieux et al. 2002Go), which may cause aberrant PKA and PrKX activities.

The protein expression patterns in human fetal brain, heart, kidney, liver, and lung are in agreement with mRNA analysis. We also compared the expression level of PrKX protein in adult and fetal human tissues. The protein level in fetal human brain was significantly higher than that in adult brain, commensurate with data obtained in mouse. In human kidney, PrKX level was higher than in adult kidney. These results agree with in situ hybridization data previously reported (Li et al. 2002Go). PrKX antigen was barely detectable in Western blots of 20-week-old human fetal heart and adult heart possibly due to earlier developmental stage expression. Abundant level of PrKX was detected in fetal human liver; in contrast, PrKX in adult liver was not detected. Together, these findings indicate that PrKX expression is age related.

In summary, partial characterization of human and murine PrKX has demonstrated a high degree of similarity as well as some notable differences with PKA including involvement in developmental processes. Our data obtained with PrKX-specific antiserum revealed characteristic patterns of PrKX expression during mouse and human development. Expression of PrKX stimulates the branching morphogenesis of MDCK cells and increases FIB4 cell migration in vitro. These effects were not observed in cells transiently expressing PKA C-subunit (Li et al. 2002Go). Growth retardation of RI mutant embryos has been observed as compared with wild-type littermates from 7.5 to 10.5 days pc (Amieux et al. 2002Go). Primary embryonic fibroblasts lacking functional RI showed abnormal cytoskeleton and migration in cell culture due to the unregulated PKA activity (Amieux et al. 2002Go). In our study, overexpression of PrKX in several epithelial cell lines was found to inhibit cell proliferation (data not shown). Thus, based on RI regulation data and the PrKX data, we hypothesize that PrKX is an important kinase involved in embryonic development.


    Footnotes
 
Received for publication November 3, 2004; accepted March 17, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Amieux PS, Howe DG, Knickerbocker H, Lee DC, Su T, Laszlo GS, Idzerda RL, et al. (2002) Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo. J Biol Chem 277:27294–27304[Abstract/Free Full Text]

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