Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit

AbstractTo explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA regionmediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation

Abnormal behaviors and developmental disorder of hippocampus in zinc finger protein 521 (ZFP521) mutant mice.

Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia

Genotype analysis of 142 offspring derived from intercrossing of <i>ZFP521</i>^+/Δ mice.

<p>From 15 <i>ZFP521</i>^+/Δ breeding pairs, 142 pups were produced. Note that the birth ratio of each genotype is consistent with Mendel's laws.</p

Organ weights of 5-week-old <i>ZFP521</i>^Δ/Δ mice.

<p>Values are presented as mean ± S.D. n = 6.</p><p>*, p<0.05 compared with <i>ZFP521^+/+</i> mice. Note that all organs of <i>ZFP521</i>^Δ/Δ mice were significantly lighter than those of <i>ZFP521</i>^+/+ mice.</p

Detection of neuroectodermal and neural stem cells.

<p>(A) Immunohistochemical detection of neuroectodermal and neural stem cells. Sections of the dentate gyrus (a–d) and cerebellum (e–h) from five-week-old <i>ZFP521^+/+</i> mouse (a, c, e, g) and <i>ZFP521</i>^Δ/Δ mouse (b, d, f, h) were stained with anti-Sox1 antibody. Note that the number of Sox1-positive cells in dentate gyrus and cerebellum of <i>ZFP521</i>^Δ/Δ mice appears smaller than that in <i>ZFP521^+/+</i> mice. Bars  = 100 μm (a, b, e, f) and 50 μm (c, d, g, h). (B) Immunoblot detection. Immunoblots of lysates from the cerebellum of five-week-old <i>ZFP521^+/+</i>, <i>ZFP521</i>^+/Δ, and <i>ZFP521</i>^Δ/Δ mice were probed with anti-Sox1 antibody (upper panel). Anti-β-actin antibody (bottom panel) was used as control for protein loading. The experiment was performed at least three times, and all results were similar. A representative result is shown. Note that Sox1 expression was decreased in the cerebellum of <i>ZFP521</i>^Δ/Δ mice. (C) Densitometric quantification of Sox1 protein in the cerebellum of five-week-old mice. The density of the Sox1 band shown above was quantified using Image-J software, and normalized by β-actin. The vertical axis indicates the quantified density of Sox1 protein relative to that in <i>ZFP521^+/+</i> mice. Error bar indicates SD. n = 6. *, P<0.001, compared to <i>ZFP521^+/+</i> mice. Note that Sox1 expression was significantly decreased in the cerebellum of <i>ZFP521</i>^Δ/Δ mice compared with <i>ZFP521^+/+</i> mice.</p

HE-staining of dentate gyrus at each developmental stage.

<p>(A) HE-staining of dentate gyrus in five-week-old mice. Bars  = 50 μm. a, <i>ZFP521^+/+</i> mouse; b, <i>ZFP521</i>^Δ/Δ mouse. Note that the density of neuronal cells was obviously lower and the neuronal cell layer was more obscure in <i>ZFP521</i>^Δ/Δ mouse. (B) HE-staining of dentate gyrus in five-day-old mice. Bars  = 100 μm (a and b) and 50 μm (c and d). a and c, <i>ZFP521^+/+</i> mouse; b and d, <i>ZFP521</i>^Δ/Δ mouse. Note that the number of neuronal cells appear smaller in five-day-old <i>ZFP521</i>^Δ/Δ mouse than in <i>ZFP521^+/+</i> mouse. (C) HE-staining of dentate gyrus and hippocampus in E19.5 embryos. Bars  = 100 μm. a, <i>ZFP521^+/+</i> embryo; b, <i>ZFP521</i>^Δ/Δ embryo. Note that the dentate gyrus and hippocampus of the <i>ZFP521</i>^Δ/Δ embryo were poorly organized, and the number of cells in the neuronal cell layer was smaller in the <i>ZFP521</i>^Δ/Δ embryo than in the <i>ZFP521^+/+</i> embryo. (D) Quantification of the number of cells in dentate gyrus. Hematoxylin positive cells were counted. Error bar indicates SD. n = 6. *, P<0.05, compared to <i>ZFP521^+/+</i> mice. Note that the number of cells in dentate gyrus of both five-week-old (5w.o) and five-day-old (P5) mice were significantly decreased compared with <i>ZFP521^+/+</i> littermates. Error bar indicates SD. n = 6.</p

Disruption of <i>ZFP521</i> gene.

<p>(A) Structure of the wild-type allele of the <i>ZFP521</i> gene, targeting vector, and recombinant allele. E, EcoRI restriction sites; closed box, location of probe used for Southern blotting (Probe A) and Northern blotting (Probe B); arrowheads a–c, PCR primers for genotyping the mice. (B) Southern blot analysis of offspring from <i>ZFP521^+/</i>^Δ mice. Genomic DNAs were digested with EcoRI, and then subjected to Southern blotting. The 7.4-kb band, recombinant allele; 6.3-kb band, wild-type allele. (C) Northern blot analysis of total mRNA. Total RNA was extracted from mouse brain, and subjected to northern blotting. Hybridization of blots with labeled cDNA probe for β-actin (middle panel), with 18S rRNA and 28S rRNA (bottom panel) used as controls for RNA loading. (D) PCR genotyping using mouse tail. Primers a and b were used for detection of the wild-type allele (412 bp), and a and c were used for the knockout allele (550 bp).</p

Histopathological analysis of the brain.

<p>(A) Photographs of the whole brains of two-week-old (a) and twenty-week-old (b) mice. The brain of <i>ZFP521</i>^Δ/Δ mouse (right brain) was smaller than that of <i>ZFP521^+/+</i> littermate (left brain). (B) HE-stained sections of cerebellum and cerebral cortex of five-week-old mice. Bars  = 100 μm. a and b, cerebellum; c and d, cerebral cortex; a and c, <i>ZFP521^+/+</i> mice, b and d, <i>ZFP521</i>^Δ/Δ mice. Note that the cerebellum and cerebral cortex of <i>ZFP521</i>^Δ/Δ mice appeared histologically normal. (C) Quantification of the number of cells in cerebellum and cerebral cortex of five-week-old mice. We counted hematoxylin positive cells, and statistically analyzed. Data represent mean ± SD. n = 5. Note that there was no significant difference in the number of cells between <i>ZFP521^+/+</i> mice and <i>ZFP521</i>^Δ/Δ mice. (D) Cortical cell layers in five-week-old mice. Sagittal sections of cerebral cortex from <i>ZFP521^+/+</i> mouse (a) and <i>ZFP521</i>^Δ/Δ mouse (b) were stained with thionin. Bars  = 100 μm. Note that the formation of six neural cell layers in the cerebral cortex of <i>ZFP521</i>^Δ/Δ mice appeared normal. (E) Immunohistochemical detection of neurons in five-week-old mice. Cerebral cortex (a and b) and cerebellum (c and d) of <i>ZFP521^+/+</i> mouse (a and c) and <i>ZFP521</i>^Δ/Δ mouse (b and d) were immunohistochemically stained with anti-NeuN antibody. Bars  = 100 μm. Note that no significant abnormality, including the number, size and positioning of differentiated neurons, was found. (F) Immunohistochemical detection of astroglial cells in five-week-old mice. Hippocampus (a and b) and cerebellum (c and d) of <i>ZFP521^+/+</i> mouse (a and c) and <i>ZFP521</i>^Δ/Δ mouse (b and d) were immunohistochemically stained with anti-GFAP antibody. Bars  = 100 μm. Note that no abnormality of astroglial cells was found. (G) Quantification of the number of anti-NeuN and anti-GFAP positive cells in cerebellum, dentate gyrus and cerebral cortex of five-week-old mice. Data represent mean ± SD. n = 6. Note that there was no significant difference in the number of cells between <i>ZFP521^+/+</i> mice and <i>ZFP521</i>^Δ/Δ mice. (H) Immunoblot detection. Immunoblots of lysates from the cerebellum of five-week-old <i>ZFP521^+/+</i> and <i>ZFP521</i>^Δ/Δ mice were probed with anti-GFAP (upper panel) and anti-NeuN antibody (middle panel). Anti-β-actin antibody (bottom panel) was used as control for protein loading. The experiment was repeated at least three times, and all results were similar. A representative result is shown. (I) Densitometric quantification of GFAP protein in the cerebellum of five-week-old mice. The density of the GFAP band shown above was quantified using Image-J software, and normalized by β-actin. The vertical axis indicates the quantified density of GFAP protein relative to that in <i>ZFP521^+/+</i> mice. Error bar indicates SD. n = 6. Note that there was no significant difference between <i>ZFP521^+/+</i> mice and <i>ZFP521</i>^Δ/Δ mice. (J) Densitometric quantification of NeuN protein in the cerebellum of five-week-old mice. The density of the NeuN band shown above was quantified using Image-J software, and normalized by β-actin. Error bar indicates SD. n = 6. Note that there was no significant difference between <i>ZFP521^+/+</i> mice and <i>ZFP521</i>^Δ/Δ mice.</p

Histological analysis of the femoral growth plate.

<p>(A) Femoral growth plate from five-week-old mice, stained with safranin O solution. The growth plate layer is strongly stained. Bars  = 100 μm (a and b) and 50 μm (c and d). a and c, <i>ZFP521^+/+</i> mouse; b and d, <i>ZFP521</i>^Δ/Δ mouse. Note that the growth plate of <i>ZFP521</i>^Δ/Δ femur appears thinner than that of <i>ZFP521</i>^+/+. (B) Histomorphometric analysis of thickness of femoral growth plate. Error bars indicate SD. n = 22. *, P<0.001. Note that the thickness of the growth plate of <i>ZFP521</i>^Δ/Δ femur is decreased by 54%, compared with that of <i>ZFP521</i>^+/+ femur.</p