TPPP/p25 Promotes Tubulin Assemblies and Blocks Mitotic Spindle Formation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 100; no. 24; pp. 13976 - 13981
• Main Authors: Tirián, L., Hlavanda, E., Oláh, J., Horváth, I., Orosz, F., Szabó, B., Kovács, J., Szabad, J., Ovádi, J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 25.11.2003; National Acad Sciences
• Subjects: Animals; Animals, Genetically Modified; Biological Sciences; Bos; Brain; Cattle; Cellular biology; Centrosomes; Drosophila; Drosophila - embryology; Drosophila - genetics; Drosophila - metabolism; Embryos; GTP; Guanosine Triphosphate - metabolism; In Vitro Techniques; Interphase; Microinjections; Microscopy, Atomic Force; Microscopy, Electron; Microtubules; Mitotic spindle apparatus; Muscle spindles; Nerve Tissue Proteins - metabolism; p25 protein; Polymerization; Proteins; Recombinant Fusion Proteins - metabolism; Spindle Apparatus - metabolism; Surface Plasmon Resonance; TPPP protein; Tubulin - metabolism; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2436331100

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:$k_{on},\>2.4 \times 10^4\>M^{-1}\!\cdot\!s^{-1};\>k_{off},\>5.4 \times 10^{-3}\>s^{-1}$; and$K_d,\>2.3 \times 10^{-7}\>M$. 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.