Profiles of PrKX Expression in Developmental Mouse Embryo and Human Tissues
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
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Summary |
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Key Words: PrKX type I cAMP-dependent protein kinase immunohistological staining Western blot
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Introduction |
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Recently, the cDNAs encoding the novel X-chromosome-encoded cAMP-dependent protein kinase, PrKX, was cloned from both human (Klink et al. 1995) and mouse (Blaschke et al. 2000
). Characterization of PrKX demonstrated that it is a type I R-subunit regulated kinase (Zimmermann et al. 1999
; Li et al. 2002
) 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 1998
; Zimmermann et al. 1999
). 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
, PKAß, and PKA
, 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. 1999
; Li et al. 2002
).
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. 1998; Schmidt et al. 2002
; Di Pasquale and Chiorini 2003
). 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 1998
; Li et al. 2002
), functional differences have also been reported (Semizarov et al. 1998
; Li et al. 2002
). Renal expression of PrKX is developmentally regulated (Li et al. 2002
). 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. 2002
). Interestingly, none of these effects was observed for PKA (Li et al. 2002
). PrKX gene expression is upregulated in HL-60 cells that have been stimulated to differentiate into granulocyte, monocyte, and macrophage lineages (Semizarov et al. 1998
; Junttila et al. 2003
). 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. 1998
; Junttila et al. 2003
).
Subcellular compartmentalization provides another level of regulation affecting the activation of the kinases by cAMP and access to substrates (Barradeau et al. 2002). Type I kinases are mainly cytoplasmic, whereas type II kinases are typically found associated with membranes and subcellular organelles (Rubin 1994
). 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 1989
). PrKX mRNA expression during mouse embryonic development and tissue distribution in adults have been analyzed using the same strategy (Blaschke et al. 2000
). 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.
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Materials and Methods |
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Characterization of the Specificity of PrKX Antibodies
PrKX and PKA were obtained from two sources for characterizing the PrKX polyclonal antibodies. PrKX and PKAC 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. 1999
). GST-PrKX and GST-PKAC
fusion proteins were expressed in yeast and purified with glutathione beads (Zimmermann et al. 1999
). The GST fusion proteins were fractionated by polyacrylamide gel electrophoresis (PAGE) with 412% 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 412% 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.
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Results |
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Discussion |
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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. 2002). 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. 2002). 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. 2002
). Primary embryonic fibroblasts lacking functional RI showed abnormal cytoskeleton and migration in cell culture due to the unregulated PKA activity (Amieux et al. 2002
). 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.
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Footnotes |
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Literature Cited |
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