Turid Holm

Phosphorylation of the Transactivation Domain of Pax6 by Extracellular Signal-regulated Kinase and p38 Mitogen-activated Protein Kinase

The transcription factor Pax6 is required for normal development of the central nervous system, the eyes, nose, and pancreas. Here we show that the transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser413) is evolutionary conserved from sea urchin to man. Ser413 is also phosphorylatedin vivo upon activation of ERK or p38 kinase. Substitution of Ser413 with alanine strongly decreased the transactivation potential of the Pax6 TAD whereas substitution with glutamate increased the transactivation. Reporter gene assays with wild-type and mutant Pax6 revealed that transactivation by the full-length Pax6 protein from paired domain-binding sites was strongly enhanced (16-fold) following co-transfection with activated p38 kinase. This enhancement was largely dependent on the Ser413 site. ERK activation, however, produced a 3-fold increase in transactivation which was partly independent of the Ser413 site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways.

Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus

A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.

The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions

The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6ΔPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein–protein interactions, but not DNA binding of Pax6ΔPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein–protein interactions.

Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations

The transcription factor Pax6 is crucial for the embryogenesis of multiple organs, including the eyes, parts of the brain and the pancreas. Mutations in one allele of PAX6 lead to eye diseases including Peters anomaly and aniridia. Here, we use fluorescence recovery after photobleaching to show that Pax6 and also other Pax family proteins display a strikingly low nuclear mobility compared to other transcriptional regulators. For Pax6, the slow mobility is largely due to the presence of two DNA-binding domains, but protein-protein interactions also contribute. Consistently, the subnuclear localization of Pax6 suggests that it interacts preferentially with chromatin-rich territories. Some aniridia-causing missense mutations in Pax6 have impaired DNA-binding affinity. Interestingly, when these mutants were analyzed by FRAP, they displayed a pronounced increased mobility compared to wild-type Pax6. Hence, our results support the conclusion that disease mutations result in proteins with impaired function because of altered DNA- and protein-interaction capabilities.

THE DEVELOPMENT OF MONOSPECIFIC ANTIBODIES AGAINST HUMAN THROMBO- PLASTIN APOPROTEIN (APOPROTEIN III) AND THEIR APPLICATION IN THE IMMUNOCYTOCHEMICAL DETECTION OF THE ANTIGEN IN BLOOD CELLS

Human thromboplastin apoprotein (apoprotein III) purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was purified a further 2-4 fold by PAGE in the presence of digitonin. Subsequent line immunoelectrophoresis of the protein revealed several lines, only one of which contained inhibitory antibodies. New inhibitory antibodies which were raised by using this particular line to immunize rabbits produced only a single line in immunoelectrophoresis of apoprotein III, with precipitated inhibitory antibodies being present only in the line. When these antibodies were used in electroblot immunobinding studies of crude thromboplastin after SDS-PAGE staining was found mainly in a single band of MW about 50,000, but also to some extent in immunologically related higher MW material. Prior deglycosylation of the thromboplastin using trinitrobenzenesulfonic acid resulted in a shift of the bulk of the main band representing an apparent MW reduction of 16%, and a corresponding shift in the position of protein with the capacity to bind inhibitory antibodies. Besides being a good criterion of specificity of the antibodies this also suggests that non-carbohydrate parts of apoprotein III may be involved in the interaction with Factor VII. Immunoperoxidase staining of unstimulated or endotoxin stimulated blood cells using the antibodies revealed the presence of significant amounts of apoprotein III only in stimulated monocytes, apparently available on the surface of the cells since it was detectable also by preembedding staining of fixed cells in suspension. The result is strong evidence that apoprotein III is synthesized de novo in monocytes upon endotoxin stimulation.

Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs.

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.