ABSTRACT
A peptide with a sequence identical to rat thymosin beta(Tb)15 was reported to be upregulated in human prostate cancer. However, in this report we provide evidence that TbNB, initially identified in human neuroblastoma, is the only Tb isoform upregulated in human prostate cancer and that the Tb15 sequence is not present herein. In addition, we demonstrate that human TbNB has a higher affinity for actin in comparison to Tb4 and promotes cell migration. In combination, this experimentally validates TbNB as functional homologue of rat Tb15 in the human organism and clarifies the current composition of the human Tb family.
Subject(s)
Prostatic Neoplasms/metabolism , Thymosin/chemistry , Thymosin/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Movement , Humans , Male , Molecular Sequence Data , Prostatic Neoplasms/genetics , RNA, Messenger/biosynthesis , Rats , Sequence Homology, Amino Acid , Thymosin/genetics , Up-RegulationABSTRACT
We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.
Subject(s)
Alternative Splicing , Contractile Proteins , Microfilament Proteins/biosynthesis , Microfilament Proteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Brain/metabolism , Dose-Response Relationship, Drug , Exons , Expressed Sequence Tags , Humans , In Situ Hybridization , Introns , Ligands , Mice , Microfilament Proteins/genetics , Molecular Sequence Data , Phosphatidylinositol 4,5-Diphosphate/pharmacology , Profilins , Protein Binding , Protein Isoforms , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Time Factors , Tissue Distribution , Transcription, GeneticABSTRACT
Profilin II dimers bind the (GP5)3 peptide derived from VASP with an affinity of approximately 0.5 microM. The resulting profilin II-peptide complex overcomes the combined capacity of thymosin beta4 and profilin II to inhibit actin nucleation and restores the extent of filament formation. We do not observe such an effect when barbed filament ends are capped. Neither can profilin I, in the presence of the peptide, promote actin polymerization during its early phase consistent with a lower affinity. Since a Pro17 peptide-profilin II complex only partially restores actin polymerization, the glycine residues in the VASP peptide appear important.
Subject(s)
Actins/chemistry , Actins/metabolism , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/metabolism , Contractile Proteins , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Amino Acid Sequence , Animals , Cattle , Dimerization , In Vitro Techniques , Microfilament Proteins/pharmacology , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Profilins , Protein Binding , Protein Conformation , Rabbits , Thymosin/pharmacologyABSTRACT
We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.