A Single cDNA Encodes Two Isoforms of Stathmin, a Developmentally Regulated Neuron-enriched Phosphoprotein*

a 19-kDa neuron-enriched soluble phos- phoprotein, as an ubiquitous intracellular for the diverse extracellular regulating cell proliferation, differentiation, Internal sequences of the protein from rat brain were determined after purification by two-dimensional polyacrylamide gel electrophoresis, electrotransfer onto Immobilon, and in situ proteolysis. Oligonucleotide mixtures based on these sequences were used to clone a cDNA for stathmin from a rat PC12 cell XgtlO library. The deduced amino acid sequence reveals partial homologies with the coiled coil structural regions of several intracellu- lar matrix phosphoproteins. Using this cDNA as a probe, we

lowing the transduction of extracellular signals (1)(2)(3)(4). A set of soluble proteins phosphorylated in response to such signals has been identified in diverse biological systems by twodimensional polyacrylamide gel electrophoresis (PAGE)' (5-7). Among these proteins, stathmin ( M , = 19,000, PI = 6.2-5.6) was recently proposed to be an ubiquitous intracellular relay for the diverse second messenger pathways activated by extracellular agents regulating the proliferation, differentiation, and functions of cells (8). It is highly abundant and a major phosphorylation substrate in neurons (9), which allowed its purification from bovine and rat brain (8,10). In the various tissues and cell types, stathmin is present as several isoelectric variants resolved by two-dimensional PAGE (8,11,12). We showed recently that they reflect the existence of two distinct isoforms 01 and P in their basal (010, PO) and increasingly phosphorylated states (010-013, PO-PS) (13).
In the present study, we cloned a cDNA for stathmin from the rat PC12 pheochromocytoma cell line which expresses both isoforms of the protein (14).* This cDNA encodes both 01 and 6 isoforms and was used to study the expression of stathmin mRNA during brain ontogenesis. Features of the deduced amino acid sequence suggest some properties which might be relevant to the molecular mechanisms underlying the biological functions of stathmin.
Stathmin Sequence Analysis-Stathmin-enriched $33 fractions were prepared by a 100 "C treatment of the rat brain soluble protein fraction (8). For sequence analysis, proteins (500 pg/gel) were run on semi-preparative two-dimensional PAGE gels (8) and electroblotted onto Immobilon membranes (17). The Amido Black-detected P1 spots were cut out from three blots, pooled, and digested with trypsin (18, 19). Peptides eluted from the membrane were purified by high pressure liquid chromatography, and the sequences of the three most abundant ones (peptides I,11,and 111,see Fig. 2) were determined by gas-phase sequencing.
Immunological Procedures-A rabbit was immunized with 2 mg of synthetic peptide I (Neosystem Laboratories, Strasbourg, France) with complete Freund's adjuvant and boosted 6 weeks later. An antibody against the entire stathmin protein was prepared previously For Western blots, proteins from gels were electroblotted onto nitrocellulose; the membrane was saturated with casein (2.5%) and probed with either antiserum as indicated. Bound antibodies were detected with 1Z51-protein A and autoradiography.
cDNA Library Screening-Two oligonucleotide mixtures (mix I and mix 11) of all the codon combinations corresponding to peptides I and I1 were synthetized (gene assembler, Pharmacia LKB Biotechnology Inc.) using deoxyinosine where codon ambiguity involved all four nucleotides. The derived 32P-end-labeledprobes (specific activity, =5 X IO6 cpm/pmol) were used to screen lo6 clones from a X g t l O tion.
of 10 clones was confirmed with mix 11. Clone XS62b that contained the longest positive EcoRI insert among four selected clones was further used for cDNA sequencing and in vitro expression. Nucleotide Sequencing-Clone XS62b insert was subcloned in both orientations into the plasmid vector pBluescript (Stratagene, San Diego, CA). Single-stranded DNA templates were prepared by infection of cultures with M13 helper phage KO7 and were sequenced by the chain termination method (21) using synthetic primers and a modified T7 DNA polymerase (Sequenase, United States Biochemical Corp., Cleveland, OH).
RNA Blot Analysis-Total RNAs were isolated from rat brain as described (22). Northern blots were prepared with 25 pg of glyoxalated RNA according to the procedure of Thomas (23) and probed with the 11-8091 EcoRi-DraI 32P-labeled (24) fragment of stathmin cDNA. Final washes were performed at 50 "C in 0.1 X SSC, 0.1% SDS. In Vitro Transcription and Translation-Linearized Bluescript recombinant plasmids pS62b were transcribed using the T3 RNA polymerase and a cap analog (Stratagene). Approximately 5 p g from the resulting cRNA were translated (2 h, 30 "C) in a rabbit reticulocyte lysate system (Promega) (25). After translation, boiled extracts were treated (1 h, 30 "C) with alkaline phosphatase (0.2 units/pl) at pH 9.5 or directly prepared for two-dimensional PAGE as described

RESULTS AND DISCUSSION
The increasingly phosphorylated states of the a and B isoforms of stathmin (aO-a3, BO-83) are partially resolved by two-dimensional PAGE as a set of unphosphorylated (Nl, N2) and phosphorylated (Pl, P2, P3) spots ( Fig. 1) (8). The amino acid sequences of three tryptic peptides (I, 11, 111) of the two-dimensional PAGE purified a1 form (spot P1) of rat brain stathmin were determined (Figs. 1 and 2). Polyclonal antibodies raised against synthetic peptide I specifically recognized all the two-dimensional PAGE spots corresponding to both a and 0 isoforms of stathmin from rat brain ( Fig. 1) or from the rat pheochromocytoma-derived PC12 cells (not shown). This result confirmed that the sequence determined is indeed that of stathmin and showed that it is common to its two isoforms.
We therefore used mixtures of synthetic oligonucleotides designed from this sequence and that of peptide I1 to screen a X g t l O PC12 cell cDNA library ( were separated by two-dimensional PAGE and silver stained or subjected to immunodetection with anti-peptide I antiserum at a 1/ 10,000 dilution (see "Experimental Procedures."). The serum recognized both stathmin isoforms (a and 8) in their unphosphorylated (a0, PO) and increasingly phosphorylated forms . N1, N2, P1, P2, and P3 designate the corresponding two-dimensional PAGE spots as they were originally identified (8). agreement with the apparent M, of 19,000 determined for stathmin by SDS-gel electrophoresis. Similarly, the 1080-base pair length of the insert containing a poly(A) tail and a 5'untranslated sequence is close to the 1.1 kilobase size of the corresponding mRNA determined by Northern blot analysis using clone XpS62b as a probe (Fig. 4A). Together, these observations indicate that clone S62b encompasses at least the nearly complete sequence of the mRNA for stathmin. The cDNA-deduced amino acid sequence contains a high proportion of charged amino acids (47%) possibly responsible for the characteristic heat stability of stathmin, which remains soluble at 100 'C (8). Furthermore, hydropathy analysis (26) revealed an increasing hydrophilicity from the N-terminal toward the C-terminal part of the molecule (not shown).
Stathmin is also rich in OH-containing residues (11 serines and 2 threonines); two of them (serines 16 and 63) are located in potential phosphorylation sites (Lys/Arg-Lys-X-Ser-X) for the CAMP-dependent protein kinase (4) for which stathmin is a good substrate (9). However, both isoforms of stathmin can be phosphorylated on at least three sites (13) and by presumably distinct kinases activated by diverse second messengers (5-7, 11, 12). Other phosphorylation sites for the CAMP-dependent and the other kinases phosphorylating stathmin in vivo remain therefore to be determined.
A search for protein sequence homology (27) revealed weak similarities between stathmin and a set of phosphoproteins of the intracellular (cytoplasmic or nuclear) matrix: myosin heavy chains (28, 29), type I1 cytokeratin (30), tropomyosin (31), and lamins A and C (32, 33) (Fig. 3). These homologies reach up to 51% for residues 47-82, within a putative predicted (3435) a-helical region of stathmin (residues 47-124). Within this region, the sequence of stathmin displays a heptad repeat structure (abcdefg)., where residues a and d are hydrophobic or uncharged and which resembles a similar organization described for the above mentioned proteins (28-33). Such heptad repeats are known to be able to yield coiled coil interacting structures (29, 32, 33, 36, 37) which might thus also contribute to the molecular mechanisms by which stath- of stathmin-enriched fractions (1 wg of protein/well) was probed with an antiserum prepared against the entire stathmin protein (8) a t a 1/ 500 dilution, and the Northern blot (b) was hybridized with clone pS62b. Both protein and mRNA levels reach a maximum at the neonatal stage. B, RNA transcribed from the clone pS62b insert was translated in vitro in a rabbit reticulocyte lysate system with ["S] methionine. A boiled extract of the lysate was then analyzed by twodimensional PAGE; the in vitro-translated proteins detected by autoradiography (b) comigrated with the silver-stained ( a ) endogenous stathmin isoforms of the reticulocyte lysate. No radioactive spot was detected when the stathmin RNA transcript was omitted from the translation system (not shown). Alkaline phosphatase treatment of the translation products (c) converted them to proteins migrating essentially as a0 and BO, the two unphosphorylated isoforms of stathmin. Less radioactive material was loaded on the gel in c than in b. min fulfills its proposed functional role as an ubiquitous intracellular relay for extracellular regulations. During brain development, the expression of stathmin protein increases until birth and then decreases toward adulthood (8, 38); it was shown by immunoprecipitation of in vitro translation products that the postnatal decrease of the protein was accompanied by a similar decline of its mRNA (38). We show by Northern blot analysis that both the increase and decrease of stathmin expression are paralleled by the expression of its mRNA (Fig. 4A). The various phases of the expression of stathmin are therefore at least in part under pretranslational and possibly transcriptional control.
T o further characterize the protein encoded by clone XS62b, in uitro-transcribed stathmin cRNA was used as a template for translation in rabbit reticulocyte lysate. As expected for stathmin (8), the translated proteins remained soluble after a 100 "C treatment used for their partial purification. In addition, when analyzed by two-dimensional PAGE, the corre-sponding [35S]methionine-labeled spots comigrated with the unphosphorylated and phosphorylated forms of the endogenous stathmin originally present in the reticulocyte lysate (Fig. 4B). Labeling experiments with [y3*P]ATP indeed demonstrated the presence of protein kinase activity in the reticulocyte lysate (not shown). Furthermore, alkaline phosphatase treatment resulted in the conversion of the in uitrotranslated proteins toward their most basic forms comigrating with the unphosphorylated states a0 and PO of the two stathmin isoforms (Fig. 4B). Altogether, these results clearly demonstrate that clone XS62b encodes proteins possessing the known biochemical properties of stathmin and whose phosphorylation takes place readily in the in vitro translation system.
The most important conclusion from the above experiments is, however, that the two a and 6 isoforms of stathmin are encoded by a single cDNA corresponding to a single cellular mRNA. Indeed, since both isoforms were expressed in the in vitro translation system, they differ only by co-translational or post-translational modifications. The amino acid following the initiator methionine being alanine, it is likely, according to Huaug et al. (39), that, in the intact cell, the methionine is cleaved and that the resulting N terminus is acetylated; these reactions are indeed known to take place also in the reticulocyte system (40). Alternate or complementary modifications on possibly different amino acid residues should, however, also be considered. Biological regulations of these post-translational reactions might then be responsible for the observed variations in the relative expression of the a and P isoforms in various tissues and cell types (8), in relation to their respective contributions to the role of stathmin as an ubiquitous intracellular relay for the regulations of cells by factors of their extracellular environment.