Exosomal TUBB3 mRNA expression of metastatic castration-resistant prostate cancer patients: Association with patient outcome under abiraterone.

BACKGROUND: To use ddPCR to quantify plasma exosomal class III ß-tubulin (ßIII-tubulin, TUBB3, encoded by the TUBB3 gene) mRNA expression in metastatic castration-resistant prostate cancer (mCRPC) patients, and study the association of this expression with abiraterone efficacy. METHODS: Blood samples were prospectively collected from 52 mCRPC patients using abiraterone as first-line therapy to measure plasma exosomal TUBB3 mRNA expression value before the initiation of abiraterone. Study endpoints were PSA response rate, PSA-progression-free survival (PSA-PFS), and overall survival (OS, from CRPC to death). RESULTS: Patients with positive exosomal TUBB3 expression showed shorter PSA-PFS (negative TUBB3 vs. positive TUBB3: 11.0 vs. 7.9 months; p = 0.014). Further analysis demonstrated that patients with strongly positive exosomal TUBB3 (>20 copies/20 µl) was associated with even shorter PSA-PFS (negative TUBB3 vs. positive TUBB3 [<20 copies/20 µl] vs. strongly positive TUBB3 [>20 copies/20 µl]: 11.0 vs. 8.3 vs. 3.6 months, p = 0.005). In multivariate analyzes, TUBB3 (+) (HR: 2.114, p = 0.033) and ECOG score >2 (HR: 3.039, p = 0.006) were independent prognosticators of poor PSA-PFS. PSA response and OS did not present significant differences. CONCLUSION: The exosomal TUBB3 mRNA expression level is associated with poor PSA-PFS of abiraterone in mCRPC patients. The detection of exosomal TUBB3 can be valuable in their management.

Identification of novel TUBB1 variants in patients with macrothrombocytopenia.

Background/aim: Macrothrombocytopenia is an autosomal-dominant disorder characterized by increased platelet size and a decreased number of circulating platelets. The membrane skeleton and the link between actin filaments of the skeleton and microtubules, which consist of alpha and beta tubulin [including the tubulin beta-1 chain (TUBB1)] heterodimers, are important for normal platelet morphology, and defects in these systems are associated with macrothrombocytopenia. Materials and methods: In this study, we sequenced the exons of the TUBB1 gene using DNA isolated from the peripheral blood samples of healthy controls (n = 47) and patients with macrothrombocytopenia (n = 37) from Turkey. The TUBB1 expression levels in fractioned blood samples from patients and healthy controls were analyzed by RT-qPCR and Western blot. Microtubule organization of the platelets in the peripheral blood smears of patients, and in mutant TUBB1-transfected HeLa cells, were analyzed by immunofluorescence staining. Results: A new TUBB1 c.803G>T (p.T178T) variant was detected in all of the control and patient samples. Importantly, we found 3 new heterozygous TUBB1 variants predicting amino acid substitutions: G146R (in 1 patient), E123Q (in 1 patient), and T274M (in 4 patients); the latter variant was associated with milder thrombocytopenia in cancer patients treated with paclitaxel. Ectopic expression of TUBB1 T274M/R307H variant in HeLa cells resulted in irregular microtubule organization. Conclusion: Further clinical and functional studies of the newly identified TUBB1 variants may offer important insights into their pathogenicity in macrothrombocytopenia.

Evaluation of α-tubulin, detyrosinated α-tubulin, and vimentin in CTCs: identification of the interaction between CTCs and blood cells through cytoskeletal elements.

BACKGROUND: Circulating tumor cells (CTCs) are the major players in the metastatic process. A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by α-tubulin (TUB), detyrosinated α-tubulin (GLU), and vimentin (VIM). In the current study, we evaluated the expression of those cytoskeletal proteins in CTCs. METHODS: Forty patients with breast cancer (BC) (16 early and 24 metastatic) were enrolled in the study. CTCs were isolated using the ISET platform and stained with the following combinations of antibodies: pancytokeratin (CK)/VIM/TUB and CK/VIM/GLU. Samples were analyzed with the ARIOL platform and confocal laser scanning microscopy. RESULTS: Fluorescence quantification revealed that the ratios CK/TUB, CK/VIM, and CK/GLU were statistically increased in MCF7 compared with more aggressive cell lines (SKBR3 and MDA-MB-231). In addition, all of these ratios were statistically increased in MCF7 cells compared with metastatic BC patients' CTCs (p = 0.0001, p = 0.0001, and p = 0.003, respectively). Interestingly, intercellular connections among CTCs and between CTCs and blood cells through cytoskeleton bridges were revealed, whereas microtentacles were increased in patients with CTC clusters. These intercellular connections were supported by TUB, VIM, and GLU. Quantification of the examined molecules revealed that the median intensity of TUB, GLU, and VIM was significantly increased in patients with metastatic BC compared with those with early disease (TUB, 62.27 vs 11.5, p = 0.0001; GLU, 6.99 vs 5.29, p = 0.029; and VIM, 8.24 vs 5.38, p = 0.0001, respectively). CONCLUSIONS: CTCs from patients with BC aggregate to each other and to blood cells through cytoskeletal protrusions, supported by VIM, TUB, and GLU. Quantification of these molecules could potentially identify CTCs related to more aggressive disease.

Biomarkers predicting chemotherapy response in head and neck squamous cell carcinoma: a review.

BACKGROUND: Biomarkers are increasingly being used in many cancers to select patients for oncological treatment paradigms based on their inherent genetic properties. However, in head and neck cancers, there are no personalised therapies available outside the context of a clinical trial. A number of studies suggest there are intrinsic tumour properties of head and neck cancers that affect their response to chemotherapeutic agents. This paper aimed to review their evidence base. METHOD: A narrative review was conducted following a search of the PubMed database. RESULTS AND CONCLUSION: The review identified a number of biomarkers predicting response to chemotherapy in head and neck cancers. The paper discusses these in detail, and explores where future research could be directed in order to deliver personalised therapies for patients with head and neck cancers.

Identification alpha-2-HS-glycoprotein precursor and tubulin beta chain as serology diagnosis biomarker of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient. Hence, the need for simple blood tests that could be used for the early detection is crucial for its ultimate control and prevention. METHODS: The present work is a case-control study focused on proteomic analysis of serum of healthy volunteers and CRC patients by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identifying a pattern of proteins/peptides able to differentiate the studied populations. Moreover, some of peptides differentially expressed in the serum of patients as compared to healthy volunteers were identified by LTQ Orbitrap XL. RESULTS: A Quick Classifier Algorithm was used to construct the peptidome patterns (m/z 1208, 1467, 1505, 1618, 1656 and 4215) for the identification of CRC from healthy volunteers with accuracy close to 100% (>CEA, P < 0.05). Peaks at m/z 1505 and 1618 were identified as alpha-2-HS-glycoprotein precursor and tubulin beta chain, respectively. CONCLUSIONS: Alpha-2-HS-glycoprotein precursor and tubulin beta chain could be involved in the pathogenesis of CRC and perform as potential serology diagnosis biomarker. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4796578761089186.

Paxillin suppresses the proliferation of HPS rat serum treated PASMCs by up-regulating the expression of cytoskeletal proteins.

Hepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation (IPVD), and arterial hypoxemia. The arterial hypoxemia induces pulmonary vascular remodelling (PVR). In recent studies, the role of the proliferation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS has been established; the changes in cytoskeletal proteins play an essential role in the proliferation of PASMCs. Little is known about the relevance of cytoskeletal protein expression or the molecular mechanisms of PVR associated with HPS. In addition, it has been identified that paxillin could influence the cytoskeletal protein expression by some important signaling pathways in many diseases, including lung cancer and liver cancer. In this study, we found that HPS rat serum from a common bile duct ligation (CBDL) rat model decreased the expression of cytoskeletal proteins (α-actin, α-tubulin, and destrin) and enhanced the expression levels of paxillin mRNA and protein in PASMCs. After silencing paxillin with siRNA, we found that the down-regulation of cytoskeletal protein expression, induced by the HPS rat serum, was reversed. Additionally, we reported that HPS rat serum improved the proliferation of PASMCs and down-regulation of paxillin could significantly inhibit this variation. These findings suggest that the up-regulation of cytoskeletal protein expression, induced by the paxillin, may cause the dysregulation of PASMC proliferation as well as play a fundamental role in PVR associated with HPS. In conclusion, down-regulation of paxillin by siRNA results in the inhibition of the dysregulation of cytoskeletal proteins and proliferation of PASMCs, suggesting a potential therapeutic effect on PVR associated with HPS.

Detection of antibodies to self-antigens (K-alpha 1 tubulin, collagen I, II, IV, and V, myosin, and vimentin) by enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting antibodies (Abs) and is employed in clinical laboratories to identify Abs against various self-antigens-autoAb development and quantitation. This method relies on specific antigen-Ab interactions where one of the components is immobilized on a solid surface. Using this method, the concentrations of antigens or Ab present in the serum can be quantified with high specificity and accuracy. Here, we describe the detection of autoAbs to various self-antigens with different tissue restriction patterns which includes collagens, k-α1 tubulin, vimentin, and myosin. We also discuss their relevance in monitoring for rejection following solid organ transplantation.

Identification of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c1 complex subunit 1 as serological diagnostic biomarkers of gastric cancer.

OBJECTIVE: Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC. DESIGN AND METHODS: The present work is a case-control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy. RESULTS: A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c1 complex subunit 1, respectively. CONCLUSIONS: The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c1 complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.

Tubulin beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex subunit 1 as serological diagnostic biomarkers of esophageal squamous cell carcinoma: a proteomics study.

Despite the major advances in diagnosis and treatment, esophageal squamous cell carcinoma (ESCC) remains a major life-threatening disease. Early diagnosis is critical for guiding the therapeutic management of ESCC. This case-control study focused on the proteomic analysis of serum of healthy volunteers and ESCC patients using the ClinProt profiling technology based on mass spectrometry. A total of 80 healthy volunteers and 119 ESCC patients were enrolled. We identified a pattern of proteins/peptides (including m/z 1867, 2700, and 2094) and differentiated ESCC patients from healthy volunteers with sensitivity and specificity close to 100%. Using mass spectrometry (LTQ orbitrap XL), tubulin beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex subunit 1 were identified as the three differentially expressed proteins/peptides in the patient serum. These three dysregulated proteins/peptides could be involved in the pathogenesis of ESCC and may serve as putative serological diagnostic biomarkers of ESCC. We suggest that further proteomics and multi-omics research are warranted to identify novel post-genomics diagnostics that can in the future pave the way for personalized medicine for patients with ESCC, a cancer for which we currently lack an integrated battery of diagnostics in the field of oncology.

ßIII-Tubulin: biomarker of taxane resistance or drug target?

INTRODUCTION: ßIII-Tubulin (TUBB3) is predominantly expressed in neurons of the central and peripheral nervous systems, while in normal non-neoplastic tissues it is barely detectable. By contrast, this cytoskeletal protein is abundant in a wide range of tumors. ßIII-Tubulin is linked to dynamic instability of microtubules (MTs), weakening the effects of agents interfering with MT polymerization. Based on this principle, early studies introduced the classical theory linking ßIII-tubulin with a mechanism of counteracting taxane activity and accordingly, prompted its investigation as a predictive biomarker of taxane resistance. AREAS COVERED: We reviewed 59 translational studies, including cohorts from lung, ovarian, breast, gastric, colorectal and various miscellaneous cancers subject to different chemotherapy regimens. EXPERT OPINION: ßIII-Tubulin functions more as a prognostic factor than as a predictor of response to chemotherapy. We believe this view can be explained by ßIII-tubulin's association with prosurvival pathways in the early steps of the metastatic process. Its prognostic response increases if combined with additional biomarkers that regulate its expression, since ßIII-tubulin can be expressed in conditions, such as estrogen exposure, unrelated to survival mechanisms and without any predictive activity. Additional avenues for therapeutic intervention could emerge if drugs are designed to directly target ßIII-tubulin and its mechanism of regulation.

Serum levels of TUBB3 correlate with clinical outcome in Chinese patients with advanced gastric cancer receiving first-line paclitaxel plus capecitabine.

The overexpression of ß-tubulin III (TUBB3) in tumor tissues was reversely related with the efficacy of paclitaxel and clinical outcome in different cancers. In this study, we aimed to investigate the association between serum levels of TUBB3 and clinical outcome in advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine. One hundred and twenty-eight advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine in Peking University Cancer Hospital from December 2006 to October 2010 were enrolled in the study. Serum samples from 32 healthy individuals were used as controls. TUBB3 expression level in advanced gastric cancer was significantly higher than that in healthy control group (31.6 ± 17.8 ng/mL vs. 16.9 ± 3.8 ng/mL, p < 0.001). For all patients, the clinical benefit rate (CBR), median progression-free survival (PFS), and overall survival (OS) were 55.6 %, 179 and 306 days, respectively. The CBR, median PFS, and OS in patients with low (n = 27) and high levels (n = 101) of TUBB3 were 95.8 %/45.1 % (low vs. high, p < 0.001), 190 days/166 days (p = 0.064), and 360 days/297 days (p = 0.023), respectively. Cox multivariate regression analysis demonstrated that the serum levels of TUBB3 were an independent prognostic factor for advanced gastric cancer patients (HR = 1.950; 95 % CI, 1.242-3.062; p = 0.004). This study indicated that low levels of TUBB3 in serum could predict better response and survival for advanced gastric cancer patients receiving paclitaxel plus capecitabine, which could be used to select patients who would benefit from this regimen.

Clinical, pharmacodynamic, and pharmacokinetic evaluation of BNC105P: a phase I trial of a novel vascular disrupting agent and inhibitor of cancer cell proliferation.

PURPOSE: To determine the recommended phase II dose and evaluate the safety and toxicity profile and pharmacokinetic (PK) and pharmacodynamic (PD) effects of BNC105P, an inhibitor of tubulin polymerization that has vascular disrupting and antiproliferative effects. EXPERIMENTAL DESIGN: BNC105P was administered as a 10-minute infusion on days 1 and 8 of a 21-day cycle in a first-in-human phase I study. A dynamic accelerated dose titration method was used for dose escalation. Plasma concentrations of BNC105P (phosphate prodrug) and BNC105 (active agent) were determined. PD assessments were carried out using dynamic contrast enhanced (DCE)-MRI and analysis of a blood-borne biomarker. RESULTS: Twenty-one subjects with advanced solid tumors were enrolled on 6 dose levels (range: 2.1-18.9 mg/m(2)). The recommended dose level was 16 mg/m(2) and was well tolerated. BNC105P (prodrug) rapidly converted to BNC105 with a half-life of 0.13 hours. Plasma concentrations of BNC105 generally increased in proportion to dose with a half-life of 0.57 hours. Pharmacodymanically active plasma levels were obtained with a dose dependant reduction in the levels of polymerized tubulin (on-target action) being observed in PBMCs. DCE-MRI also indicated blood flow changes in the tumor lesions of a number of subjects. CONCLUSIONS: BNC105P has a favorable toxicity profile at the recommended dose of 16 mg/m(2) and is associated with PD changes consistent with its known mechanism of action. Phase II studies in renal cancer and mesothelioma have commenced.

Phase II study of paclitaxel and vinorelbine (Pacl-Vin) in hormone-refractory metastatic prostate cancer: double tubulin targeting.

BACKGROUND: Androgen ablation is the standard treatment for advanced prostate cancer. However, most patients will eventually develop progressive hormone-refractory prostate cancer (HRPC). The aim of the Pacl-Vin study was to determine the efficacy and safety of paclitaxel in combination with vinorelbine in patients with HRPC, following from a phase I trial. METHODS: Thirty castrate patients with progressive, metastatic prostate cancer were enrolled. Patients were treated with paclitaxel 40 mg/m2, vinorelbine 20 mg/m2 intravenously on day 1 and day 8 of a 21-day cycle. RESULTS: Two patients demonstrated a partial response and seven patients had stable disease from a cohort of 10 patients with measurable disease. Of 30 patients assessable for prostate-specific antigen (PSA) response, 19 showed stable disease, which was maintained for at least 4 weeks, while six (20%) experienced>or=50% decline in PSA levels. Median overall survival was 7.3 months (interquartile range (IQR): 4.7-9.9 months). Median progression-free survival was 3.3 months (IQR: 2.5-7.0 months). Improvement in quality of life measures was noted after three cycles of therapy. Grade 3 and 4 toxicities were: neutropenia 8%, febrile neutropenia 4%, infection 2%, anaemia 3%, lethargy 1% and somnolescence 1%. One patient died as a result of neutropenic sepsis. CONCLUSION: In a poor prognostic cohort of patients paclitaxel and vinorelbine is a tolerable regimen, with a 20% PSA and objective response rate. The majority of patients achieved PSA stability. Furthermore, quality of life parameters, such as pain, were improved. However, the low level of activity of this regimen precludes its further testing.

Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

ADPRibosylation of chicken red cell tubulin and inhibition of microtubule self-assembly in vitro by the NAD(+)-dependent avian ADPRibosyl transferase.

Chicken erythrocyte tubulin was found to undergo NAD(+)-dependent ADPribosylation in vitro in the presence of ADPRtransferase also isolated from avian red blood cells. Unlike the low level of ADPR incorporation catalyzed by Cholera and Pertussis toxins (i.e., less than 0.005 mol ADPR/mol tubulin), the avian system displayed a much higher stoichiometry of 0.8-1.2 mol ADPR/mol tubulin. Modification resulted in potent inhibition of microtubule self-assembly, even in the presence of bovine brain microtubule-associated proteins or with the addition of pre-assembled microtubules.

The role of microtubules in platelet secretory release.

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.

Time resolved analysis of tubulin phosphorylation during platelet activation.

Tubulin phosphorylation was analyzed during the different phases of platelet activation. Platelets preloaded with [32P]-phosphate were stimulated with collagen. Tubulin was immunoprecipitated from serial samples obtained during the activation process. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and autoradiographs analyzed by laser densitometry. Agonist induced dephosphorylation of platelets occurred after the onset of shape change at the time of initiation of the secretory release. The dephosphorylation was selective affecting specific peptides.

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.