Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

BACKGROUND: Two main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

<p>Abstract</p> <p>Background</p> <p>Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits.</p> <p>Results</p> <p>We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level.</p> <p>Conclusion</p> <p>Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.</p

Chronic Lymphocytic Leukemia Cells Are Activated and Proliferate in Response to Specific T Helper Cells

There is increasing interest in the chronic lymphocytic leukemia (CLL) microenvironment and the mechanisms that may promote CLL cell survival and proliferation. A role for T helper (Th) cells has been suggested, but current evidence is only circumstantial. Here we show that CLL patients had memory Th cells that were specific for endogenous CLL antigens. These Th cells activated autologous CLL cell proliferation in vitro and in human → mouse xenograft experiments. Moreover, CLL cells were efficient antigen-presenting cells that could endocytose and process complex proteins through antigen uptake pathways, including the B cell receptor. Activation of CLL cells by Th cells was contact and CD40L dependent. The results suggest that CLL is driven by ongoing immune responses related to Th cell–CLL cell interaction. We propose that Th cells support malignant B cells and that they could be targeted in the treatment of CLL

(a) Comparison of liver from wild type and PKA Cα and Cβ ablated mice

PKA Cα KO mice showed a clear uniform reduction in size. (b) Confocal immunofluorescence microscopy of frozen liver sections stained by sheep anti-EGFR and Cy3-conjugated donkey anti-sheep antibodies. (c) Western immunoblotting analysis of EGFR expression in liver and brain from wt (+/+), heterozygote (-/+), and Cα and Cβ KO (-/-) mice. Immunoblots were incubated with sheep anti-EGFR, rabbit anti-pan PKA C and mouse anti-PKA C, and rabbit anti-erbB2. Secondary HRP-conjugated anti-IgG antibodies were used for detection. (d) PKA kinase activity in mouse liver. Activity was assayed by phosphorylation of the PKA-specific substrate Kemptide using γ-[P]ATP. The assay was performed in the presence of cAMP. Activity was measured by liquid scintillation in 3 ml Opti-fluor. Values are given as counts per minute (cpm).<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit"</p><p>http://www.biomedcentral.com/1471-2121/9/16</p><p>BMC Cell Biology 2008;9():16-16.</p><p>Published online 1 Apr 2008</p><p>PMCID:PMC2324083.</p><p></p

(a) HeLa cells incubated with RNAi against Cα and Cβ and monitored for PKA-specific phosphotransferase activity in cell extracts of 1 mg/mL

Relative values given as counts per minute (cpm). (b) Relative EGFR mRNA expression levels were measured by RT-PCR and are given as E/G × 100 where E and G are the relative copy numbers of EGFR and GAPDH mRNA, respectively. The results are given as +/- SEM where n = 3. (c) Western immunoblotting analysis of expression of EGFR and PKA C. An anti-actin antibody was applied to ensure equal loading.<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit"</p><p>http://www.biomedcentral.com/1471-2121/9/16</p><p>BMC Cell Biology 2008;9():16-16.</p><p>Published online 1 Apr 2008</p><p>PMCID:PMC2324083.</p><p></p

Linear regression analysis showed that both Cα and Cβ KO mice have a significantly lower body weight when compared to WT mice (P 0,0001) when adjusted for age

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit"</p><p>http://www.biomedcentral.com/1471-2121/9/16</p><p>BMC Cell Biology 2008;9():16-16.</p><p>Published online 1 Apr 2008</p><p>PMCID:PMC2324083.</p><p></p