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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 (⌬3-43TPPP) 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. Previously we isolated a new, flexible, intrinsically unstructured protein from bovine brain, which was denoted tubulin polymerization-promoting protein (TPPP) 3 /p25 accordingly to its in vitro function, tubulin polymerizationpromoting protein, and its molecular mass (1). In the HGNC data base this protein is assigned as TPPP, the first member of TPPP family. We have shown that TPPP can induce formation of double-walled microtubules (MTs) and aberrant tubulin aggregates (2) as well as that it stabilizes MT network via its bundling activity in human cells (3). There are two TPPP homologues, p18 (TPPP2) and p20 (TPPP3) with distinct structural and functional features concerning their folding and tubulin binding properties (4). The physiological functions of the TPPP protein family are unknown; however, TPPP has been suggested to take part in the stabilization of the MT network (3).Both ␣-synuclein and GAPDH directly bind to TPPP and co-localize in the Lewy body (5-7). Our immunohistochemistry studies revealed enrichment of TPPP in ␣-synuclein-positive inclusions characteristic for synucleinopathies but not for tauopathies (5). TPPP is similar to ␣-synuclein and tau (2,8) as far as all of them belong to the family of the unstructured proteins.Protein phosphorylation has a significant impact in the etiology of neurodegenerative processes (9). For example, tau and ␣-synuclein, the major hallmarks of Parkinson and Alzheimer diseases, respectively, are targets of different protein kinases. One of the kinases phosphorylating tau with pathological relevance in Alzheimer disease is cyclin-dependent kinase 5 (Cdk5), which is abundant in brain tissue, associates with tau (10), and plays important role in the signaling of mitogen-activated protein kinases ...

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