TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.
Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast. Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 x 10(4) M(-1) x s(-1); koff, 5.4 x 10(-3) s(-1); and Kd, 2.3 x 10(-7) M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin-GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo.
Tirián
L
L
Department of Biology, Faculty of Medicine, University of Szeged, H-6720, Szeged, Hungary.
Hlavanda
E
E
Oláh
J
J
Horváth
I
I
Orosz
F
F
Szabó
B
B
Kovács
J
J
Szabad
J
J
Ovádi
J
J
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D008845:Microinjections; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014404:Tubulin / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate