A novel partner for Dictyostelium filamin is an alpha-helical developmentally regulated protein.
The filamins are a family of highly homologous actin-crosslinking proteins that stabilize three-dimensional actin networks, link them to membrane proteins and direct intracellular signaling reactions to the actin scaffold through interaction with various binding partners. Here, we describe the first Dictyostelium filamin-interacting protein to be isolated--FIP, a 229.8 kDa protein with two alpha-helical coiled coil domains. FIP was identified in a yeast two-hybrid screen using the rod domain of filamin as bait. FIP can also be coimmunoprecipitated with filamin from cellular extracts. Deletion analysis located the interaction domain of FIP to a C-terminal region; by contrast, in filamin rods, repeats 2-4 interacted with the recombinant FIP protein. The 7 kb transcript of FIP is upregulated during early development. Monoclonal antibodies raised against a bacterially expressed FIP polypeptide recognize a 230 kDa developmentally regulated protein in western blots. Immunofluorescence analysis shows a punctate staining pattern in the cytosol and, in cell fractionation experiments, FIP is mainly found in the cytosolic fraction. A fusion protein composed of GFP and the C-terminal part localizes to the plasma membrane and is associated with the cytoskeleton. Expression of the fusion protein affects development and influences the size of the multicellular aggregates and the phototactic behavior of slugs. Thus, FIP might provide a candidate link between the dynamic actin cytoskeleton and signal transduction events during the multicellular stages of Dictyostelium amoebae.
Knuth
Monika
M
Zentrum Biochemie, Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, 50931, Germany.
Khaire
Nandkumar
N
Kuspa
Adam
A
Lu
Si Jie
SJ
Schleicher
Michael
M
Noegel
Angelika A
AA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D064448:Filamins; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D008027:Light; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003285:Contractile Proteins; D018076:DNA, Complementary; D064448:Filamins; D008840:Microfilament Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins