Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions

Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin-integrin complexes into fibronectin-mediated fibrillar adhesions.     

Expression,purification and characterisation of a novel mutant of the human protein kinase CK2

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant subunit. According to our theoretical predictions, we observed that the -helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.     

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.