Journal: Cancer Research

Article Title: Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

doi: 10.1158/0008-5472.can-11-2520

Figure Lengend Snippet: Figure 3. Phosphorylation and dephosphorylation of Thr443. A, in vitro kinase assay was carried out with the CS-PKA on GST fusion proteins containing the IC domain of CA IX in the wt version and in the mutated version with Thr443 replaced by Ala (T443 ! A). The upper part of the figure represents a CBB-stained gel (loading control), the lower part represents autoradiograph of the proteins phosphorylated in the presence of [g-32P]ATP. B, in vitro phosphatase assay was carried out with heterodimeric and heterotrimeric forms of PP2A on PKA-phosphorylated GST-tagged wt-IC fragment of CA IX. The upper part of the figure represents a gel with CBB-stained substrate proteins (loading control), the lower part represents autoradiograph. The data show that Thr443 is a substrate residue for PKA and PP2A. The experiments (in A and B) were repeated twice and representative results are shown. C, CA IX was immunoprecipitated by the M75 MAb from cell extracts, in vitro phosphorylated by CS-PKA, subjected to PAGE, blotted and incubated with the P-T443 CA IX antibody. In vitro phosphorylated GST-tagged wt-IC domain of CA IX protein (GST-IC) was used as a positive control. D, all 3 blots contain PAGE-resolved extracts from HEK293T/17 cells transfected with different combinations of the following plasmids: pcDNA3.1 (p1), pSG5C (p2), pSG5C-MN/CA9 (pCA9), pcDNA3.1- CSPKA (pCa), pcDNA3.1dnPKA (pdn). The plasmids were cotransfected in a ratio of 10:1. The blots were incubated with antibodies as indicated on the left side. The data show that phosphorylation of CA IX, activation of PKA and phosphorylation of PKA substrates occur only in the presence of CS-PKA. The experiment was repeated 4 times.

Article Snippet: HEK293T/17 cells were cotransfectedwith various combinations of the following plasmids: pCalpha-EV-PKA encoding CSPKA (pCa, catalytic subunit C alpha, 15,310 Addgene plasmid; ref. 17), pcDNA3.1þdnPKA encoding DN-PKA (pdn), pSG5CMN/CA9 encoding CA IX (pCA9) and respective empty plasmidsbyNeoFectin in the ratio10:1, 4mgofplasmidDNA in total.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, In Vitro, Kinase Assay, Staining, Control, Autoradiography, Phosphatase Assay, Residue, Immunoprecipitation, Incubation, Positive Control, Transfection, Activation Assay

Journal:

Article Title: Cyclic AMP-Dependent Protein Kinase Catalytic Subunits Have Divergent Roles in Virulence Factor Production in Two Varieties of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1128/EC.3.1.14-26.2004

Figure Lengend Snippet: Two C. neoformans genes encode PKA catalytic subunits. (A) PKA catalytic subunits identified in C. neoformans were aligned with the Uka1 protein from the related basidiomycete fungal pathogen U. maydis with the CLUSTALW feature of MacVector (Oxford Molecular). Conserved residues are boxed, with identities shown in bold letters and darkly shaded boxes, and similarities are indicated by lightly shaded boxes. (B) The evolutionary relationships between the PKA catalytic subunits of C. neoformans, U. maydis, S. cerevisiae, A. nidulans, and C. albicans were compared by using the neighbor-joining function of the ClustalX program (62). A phylogenetic tree was then constructed with the TreeView program (46). The proteins are positioned on the tree according to sequence similarity. Evolutionary dissimilarity is proportional to the horizontal distance joining any two proteins on the tree.

Article Snippet: Inter- and intravarietal comparisons of the PKA catalytic subunits revealed that while the serotype A Pka1 and Pka2 proteins share 93% identity with their respective counterparts in serotype D, there is only 33 to 35% identity shared between the Pka1 and Pka2 subunits, either within or between varieties. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 1. caption a7 Two C. neoformans genes encode PKA catalytic subunits. (A) PKA catalytic subunits identified in C. neoformans were aligned with the Uka1 protein from the related basidiomycete fungal pathogen U. maydis with the CLUSTALW feature of MacVector (Oxford Molecular).

Techniques: Construct, Sequencing