title: Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases.
abstract: Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.
authors: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, H-1113, Hungary.
authors: Emma
authors: E
authors: Hlavanda
authors: Eva
authors: E
authors: Klement
authors: Endre
authors: E
authors: Kókai
authors: János
authors: J
authors: Kovács
authors: Orsolya
authors: O
authors: Vincze
authors: Natália
authors: N
authors: Tökési
authors: Ferenc
authors: F
authors: Orosz
authors: Katalin F
authors: KF
authors: Medzihradszky
authors: Viktor
authors: V
authors: Dombrádi
authors: Judit
authors: J
authors: Ovádi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C118136:TPPP protein, human; D012694:Serine; D011494:Protein Kinases; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D019950:Mitogen-Activated Protein Kinase 1