Identification by mass spectrometry of a new alpha-tubulin isotype expressed in human breast and lung carcinoma cell lines.

The extensive C-terminal molecular heterogeneity of alpha- and beta-tubulin is a consequence of multiple isotypes, the products of distinct genes, that undergo several posttranslational modifications. These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha-tubulin, and phosphorylation of the beta III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human cell lines. Total cell extracts are resolved by SDS--PAGE and transferred to nitrocellulose, and the region of the blot corresponding to tubulin (approximately 50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human alpha- and beta-tubulin CNBr-derived C-terminal peptides are all in the 1500--4000 Da mass range and can be analyzed directly by MALDI-TOF mass spectrometry in the negative ion mode without significant interference from other released peptides. In this study, the tubulin isotype diversity in MDA-MB-231, a human breast carcinoma cell line, and A549, a human non-small lung cancer cell line, is reported. The major tubulin isotypes present in both cell lines are k-alpha 1 and beta 1. Importantly, we report a previously unknown alpha isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of alpha-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-alpha 1 and beta 1 isotypes was observed in these two human cell lines.

A common pharmacophore for Taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain.

Extensive structure-activity studies done with Taxol have identified the side chain at C-13 as one of the requirements for biological activity. Baccatin III, an analogue of Taxol lacking the C-13 side chain, has none of the biological characteristics of Taxol. Since 2-m-azido Taxol, a Taxol derivative with a m-azido substituent in the C-2 benzoyl ring, has greater activity than Taxol, we questioned whether 2-m-azido baccatin III might be active. 2-m-Azido baccatin III inhibited the proliferation of human cancer cells at nanomolar concentrations, blocked cells at mitosis, and reorganized the interphase microtubules into distinct bundles, a typical morphological change induced by Taxol. In contrast to 2-m-azido baccatin III, 2-p-azido baccatin III was similar to baccatin III, having no Taxol-like activity, further indicating the specificity and significance of the 2-meta position substituent. Molecular modeling studies done with the C-2 benzoyl ring of Taxol indicated that it fits into a pocket formed by His227 and Asp224 on beta-tubulin and that the 2-m-azido, in contrast to the 2-p-azido substituent, is capable of enhancing the interaction between the benzoyl group and the side chain of Asp224. The observation that the C-13 side chain is not an absolute requirement for biological activity in a taxane molecule has enabled the development of a new common pharmacophore model between Taxol and the epothilones.

Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology.

We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1alpha and TcPP1beta. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the T. cruzi genome. The complete coding region for TcPP1beta was expressed in Escherichia coli by using a vector, pTACTAC, with the trp-lac hybrid promoter. The recombinant protein from the TcPP1beta construct displayed phosphatase activity toward phosphorylase a, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC(50)], approximately 2 nM) over okadaic acid (IC(50), approximately 100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 microM did not result in growth arrest or morphological alterations to T. cruzi epimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of (32)P-labeled phosphorylase a by T. cruzi epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.

Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1.

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.

Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol.

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.

Developmental expression of the protein kinase C substrate/binding protein (clone 72/SSeCKS) in rat testis identification as a scaffolding protein containing an A-kinase-anchoring domain which is expressed during late-stage spermatogenesis.

The coordinated interaction of kinases, phosphatases and other regulatory molecules with scaffolding proteins is emerging as a major theme in intracellular signaling networks. In this report we show that a cDNA isolated from a rat testis expression library by interactive cloning using the regulatory subunit (R) of a type-II protein kinase A (PKA) is identical with a previously characterized protein kinase C (PKC)-binding protein termed either clone 72 [Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C. & Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422] or SSeCKS [Lin, X., Tombler, E., B., Nelson, P.J., Ross, M. & Gelman, I.H. (1996) J. Biol. Chem. 271, 28430-28438]. Deletion mutagenesis demonstrated that amino acids 1495-1524 of clone 72/SSeCKS had the ability to interact with RII. Antibodies prepared against the recombinant protein recognized a 280/290-kDa doublet and a 240-kDa protein on Western blots of rat testis cytosolic and Triton X-100 extracts. Expression of clone 72/SSeCKS mRNA and protein levels was developmentally regulated in rat testis. Northern-blot analysis showed a dramatic increase in clone 72/SSeCKS-hybridizing mRNA starting 30 days after birth. Immunohistochemical examination showed high expression levels in elongating spermatids. Clone 72/SSeCKS was not detected in mature sperm. These studies suggest a role for clone 72/SSeCKS, a PKA/PKC scaffolding protein, during the process of spermiogenesis.

Polar tube proteins of microsporidia of the family encephalitozoonidae.

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem, a microsporidian parasite of humans.

The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Identification of the domains of photoincorporation of the 3'- and 7-benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein.

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic compounds across the plasma membrane. The transporter is composed of two homologous halves, each containing a nucleotide binding fold and six putative transmembrane spanning segments. The contact domains between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, the CNBr fragment generated from the 7-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab4) raised against amino acid residues 740-750. The specificity of these reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.

Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit.

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.

Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm.

cAMP is important for the initiation of mammalian sperm motility. Previously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum. Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII. An RII-binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa. In this study, we report the cloning of a rat testis-specific, developmentally regulated, RII-binding protein (TAKAP-80). A 1.2-kb cDNA clone, isolated by screening a rat testis expression library with 32P-labeled RII, hybridized to a 1.8-kb mRNA transcript present exclusively in testis. This transcript appeared at detectable levels at 30 days after birth. Over the next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleotide sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1-147 of the protein contained 45% charged residues, with lysine and arginine predominating. Similarly, amino acids 268-502 also contained a high percentage of charged amino acids (35%). In contrast, amino acids 148-267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2-kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli. The recombinant protein was soluble and bound RIIalpha, RIIbeta and type IIalpha holoenzyme by the RII-binding overlay procedure. Deletion analysis revealed that the high-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80. Antibodies prepared against the fusion protein recognized an 80-kDa protein present in the urea-insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm. Levels of the 80-kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.

Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein.

P-glycoprotein, the multidrug resistance transporter, is phosphorylated in vivo and the major phosphorylation domain has been identified as the linker region (amino acids 629-686). The linker region is a highly charged segment of the transporter in which the negative and positive amino acid side chains are spatially segregated. Both of these charged domains contain several consensus phosphorylation sites for protein kinases. Three of the consensus phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein are utilized in vivo and have been identified as serines 665, 669, and 681. Mutagenesis of all the consensus basic-directed kinase phosphorylation sites in the linker region of human MDR1 P-glycoprotein did not alter the ability of the mutated transporter to confer the multidrug resistance phenotype in stably transfected cell lines. These studies would suggest that phosphorylation/dephosphorylation within the basic domain of the linker region is not directly involved in regulation of drug transporter activity. We now report that the linker region of mdr1b P-glycoprotein is also phosphorylated in vivo within the acidic domain (amino acids 631-658). These sites have been mapped using casein kinase II, a prototypic acidic-directed kinase, and a recombinant mdr1b linker region peptide (amino acids 621-687). Electrospray mass spectrometry demonstrated that casein kinase II could introduce up to five phosphates into the recombinant peptide. Two-dimensional phosphopeptide mapping indicated that all the phosphates were contained in a tryptic peptide consisting of amino acids 631-658. Phosphopeptide mapping of in vivo labeled P-glycoprotein, isolated from either J7.V1-1, a murine vinblastine-resistant cell line, or HeLa cells stably transfected with mdr1b P-glycoprotein cDNA, revealed that this tryptic peptide was phosphorylated in both proteins.

Domain organization of murine mdr1b P-glycoprotein: the cytoplasmic linker region is a potential dimerization domain.

P-glycoprotein is an integral membrane protein that functions as a cytotoxic drug-efflux pump. Studies suggest that the transporter exists in the membrane as a dimer and possibly higher order structures. We report the bacterial expression of the linker region (amino acids 621-688) of murine mdr1b P-glycoprotein and demonstrate that this region, which serves to link the two homologous halves of the transporter, has the potential to serve as a dimerization domain. The recombinant peptide (8742 daltons) eluted from a gel filtration column at a position corresponding to a dimer (i.e. 17,500 daltons). A dimer:monomer equilibrium, as a function of peptide concentration, was confirmed by large zone gel filtration chromatography. The dissociation constant (KD) for the dimer:monomer equilibrium was 350 nM. It was possible to dissociate the dimer by low pH (3.0) or high ionic strength (2.5 M NaCl). Dimerization was not affected by an alkaline pH of 10 or 5 mM EDTA. Studies with a truncated linker peptide indicated that the N-terminal 48 amino acids were sufficient for dimerization.

Characterization of the taxol binding site on the microtubule. 2-(m-Azidobenzoyl)taxol photolabels a peptide (amino acids 217-231) of beta-tubulin.

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N.E., Heerding, J.M., Swindell, C.S., Ringel, I., Orr, G.A., and Horwitz, S.B. (1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels beta-tubulin and the photolabeling is completed by both taxol and unlabeled 2-(m-azidobenzoyl)-taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of beta-tubulin as the major photolabeled domain.