• BMB Reports
• /
• v.34 no.1
• /
• pp.33-38
• /
• 2001

The human PTK6 (also known as Brk) polypeptide, which is deduced from its full-length cDNA, represents a non-receptor protein tyrosine kinase (PTK). It contains SH3, SH2, and tyrosine kinase catalytic domains that are closely related to Src family members. We generated an antihuman PTK6 antibody by immunizing rabbits with a PTK6-specific oligopeptide conjugated to BSA, which corresponds to 11 amino acid residues near the C-terminus. An immunoblot analysis with the antibody detected an expected 52-kDa band in various mammalian transformed cell lines. Immunoprecipitation and immunoblot analyses demonstrated that PTK6 is phosphorylated on the tyrosine residues) and interacts with approximately a 23-kDa tyrosine-phosphorylated polypeptide (most likely a substrate of PTK6) in breast carcinoma T-47D cells. An immunofluorescence analysis demonstrated that PTK6 is localized throughout the cytoplasm of T-47D cells. These results support a possible role for PTK6 in the intracellular signal transduction through tyrosine phosphorylation.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.5
• /
• pp.1043-1046
• /
• 2009

Human protein tyrosine kinase-6 (PTK6) is a member of the non-receptor protein tyrosine kinase family and it is found in two-thirds of all breast tumors. Very recently, we proposed that the SH3 domain of PTK6 interacts with the linker region (Linker) between the SH2 and kinase domains, proving that the interaction between SH3 domain and Linker plays an important role in auto-inhibition mechanism. Residues from 1 to 191 corresponding region of SH3-SH2-Linker (SH32L) of PTK6 was cloned into the pET32a expression vector with Tobbaco etch virus (TEV) protease enzyme site by sequence homology and 3D structural model. The purified PTK6-SH32L was determined as a monomer conformation in solution. The amide proton resonances in the $^{15}N-^{1}H$ 2D-HSQC spectrum suggest that PTK6-SH32L possesses disordered structural region of the flexible/unstructured linker region. In addition, the backbone amide proton chemical shifts of the SH3 domain in the PTK6-SH32L differ from that of the independent domain, indicating that intra-molecular interaction between SH3 and Linker in the PTK6-SH32L is present.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.179-184
• /
• 2007

PTK6, an intracellular protein tyrosine kinase, is significantly overexpressed in a majority of breast cancers and has a role in promoting the proliferation of the cancer cells, but not of normal cells. Here, we report high-level production of the catalytic unit of PTK6 fused with Drosophila peptidoglycan recognition protein (PGRT)-LB, in the baculovirus system. We first found that the PGRP-LB was potentially useful as a fusion partner to increase the yield of heterologous protein in the baculovirus system. The purified recombinant protein exhibited a 1.5-fold activity with much higher yield than the bacterially-expressed protein. The protein expressed in the baculovirus system will be useful for the crystallization to determine its crystal structure helping understand the molecular mechanism of PTK6 and design its inhibitors.

• BMB Reports
• /
• v.35 no.3
• /
• pp.343-347
• /
• 2002

The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase. We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques. The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of $\beta$-sheet conformations. It is most stable when the pH is neutral based on the pH titration data. In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition. For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments. Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.

• Journal of Korean Neurosurgical Society
• /
• v.65 no.1
• /
• pp.64-73
• /
• 2022

Objective : To evaluate the surgical outcomes of partial pedicle subtraction osteotomy (PPSO) in patients with thoracolumbar fractures and compare the outcomes of PPSO for burst fractures with those for posttraumatic kyphosis (PTK). Methods : From June 2013 to May 2019, 20 consecutive adult patients underwent PPSO for thoracolumbar fractures at the levels of T10 to L2. Of these patients, 10 underwent surgery for acute fractures (burst fractures), and 10 for sequelae of thoracolumbar fractures (PTK). Outcomes of PPSO were evaluated and compared between the groups. Results : Twenty patients (each 10 patients of burst fractures and PTK) with a mean age of 64.7±11.1 years were included. The mean follow-up period was 21.8±11.0 months. The mean correction of the thoracolumbar angle was -34.9°±18.1° (from 37.8°±20.5°preoperatively to 2.8°±15.2° postoperatively). The mean angular correction at the PPSO site was -38.4°±13.6° (from 35.5°±13.6° preoperatively to -2.9°±14.1° postoperatively). The mean preoperative sagittal vertical axis was 93.5±6.7 cm, which was improved to 37.6±35.0 cm postoperatively. The mean preoperative kyphotic angle at the PPSO site was significant greater in patients with PTK (44.8°±7.2°) than in patients with burst fractures (26.2°±12.2°, p=0.00). However, the mean postoperative PPSO angle did not differ between the two groups (-5.9°±15.7° in patients with burst fractures and 0.2°±12.4° in those with PTK, p=0.28). The mean angular correction at the PPSO site was significantly greater in patients with PTK (-44.6°±10.7°) than in those with burst fractures (-32.1°±13.7°, p=0.04). The mean operation time was 188.1±37.6 minutes, and the mean amount of surgical bleeding was 1030.0±533.2 mL. There were seven cases of perioperative complications occurred in five patients (25%), including one case (5%) of neurological deficit. The operation time, surgical bleeding, and complication rates did not differ between groups. Conclusion : In cases of burst fracture, PPSO provided enough spinal cord decompression without corpectomy and produced sagittal correction superior to that achieved with corpectomy. In case of PTK, PPSO achieved satisfactory curve correction comparable to that achieved with conventional PSO, with less surgical time, less blood loss, and lower complication rates. PPSO could be a viable surgical option for both burst fractures and PTK.

• Journal of Korean Neurosurgical Society
• /
• v.65 no.1
• /
• pp.84-95
• /
• 2022

Objective : To investigate the clinical efficacy and safety of the controlled distraction-compression technique using an expandable titanium cage (ETC) in posttraumatic kyphosis (PTK). Methods : We retrospectively studied and collected data on 20 patients with PTK. From January 2014 to December 2017, the controlled distraction-compression technique using ETC was consecutively performed in 20 patients with PTK of the thoracolumbar zone (range, 36-82 years). Among them, nine were males and 11 were females and the mean age was 61.5 years. The patients were followed regularly at 1, 3, 6, and 12 months, and the last follow-up was more than 2 years after surgery. Results : The mean follow-up period was 27.3±7.3 months (range, 14-48). The average operation time was 286.8±33.1 minutes (range, 225-365). The preoperative regional kyphotic angle (RKA) ranged from 35.6° to 70.6° with an average of 47.5°±8.1°. The immediate postoperative mean RKA was 5.9°±3.8° (86.2% correction rate, p=0.000), and at the last follow-up more than 2 years later, the mean RKA was 9.2°±4.9° (80.2% correction rate, p=0.000). The preoperative mean thoracolumbar kyphosis was 49.1°±9.2° and was corrected to an average of 8.8°±5.3° immediately after surgery (p=0.000). At the last follow-up, a correction of 11.9°±6.3° was obtained (p=0.000). The preoperative mean back visual analog scale (VAS) score was 7.9±0.8 and at the last follow-up, the VAS score was improved to a mean of 2.3±1.0 with a 70.9% correction rate (p=0.000). The preoperative mean Oswestry disability index (ODI) score was 32.3±6.9 (64.6%) and the last follow-up ODI score was improved to a mean of 6.85±2.9 (3.7%) with a 78.8% correction rate (p=0.000). The overall complication was 15%, with two of distal junctional fractures and one of proximal junctional kyphosis and screw loosening. However, there were no complications directly related to the operation. Conclusion : Posterior vertebral column resection through the controlled distraction-compression technique using ETC showed safe and good results in terms of complications, and clinical and radiologic outcomes in PTK. However, to further evaluate the efficacy of this surgical procedure, more patients need long-term follow-up and there is a need to apply it to other diseases.

• Radiation Oncology Journal
• /
• v.18 no.1
• /
• pp.51-58
• /
• 2000

Purpose :The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive KS62 leukemia cell line was investigated. Materials and Methods :K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2×106 cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37$^{\circ}C$ for 0$\~$48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bel-2, bel-X$_{L}$ and bax protein levels. Results :Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electro-phoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bel-2 or bel-X$_{L}$ anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30$\~$40$\%$ at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. Conclusion : We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210$^{bcr/abl}$ failed to enhance the radiation induced apoptosis in KS62 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bel-2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation-induced apoptosis.

• BMB Reports
• /
• v.30 no.6
• /
• pp.431-437
• /
• 1997

Since the role of CD40 on the interleukin-4(IL-4) -induced B cell activation has been strongly implicated in the agumentation of IgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type II IgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti -CD40-induced CD23 expression. whereas protein kinase C (PKC) inhibitors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and $NF-_{K}B$, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors. each of which is rapidly activated via PKC-independent and PTK-dependent process.

• Radiation Oncology Journal
• /
• v.21 no.3
• /
• pp.227-237
• /
• 2003

Purpose: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HWA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosils on $p210^{bcr/abl}$ protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the Induction on a number of transcription factors and the differential gene expression in this model were investigated. Materials and Methids: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 Mev Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with $0.25/muM$ of HMA and $25/muM$ of genistein, and the expressions and the activities of abl kinase, MAPK family, NF- kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. Results: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either in association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF- kB activity and the TK1 expression and activity. Conclusion: The effects of HMA and genistein on the radiosensitivity on the K562 cells were not related to the bcr-abl kinase activity in this study, another signaling pathway, besides the WAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.

• Proceedings of the Korean Magnetic Resonance Society Conference
• /
• 2000.08a
• /
• pp.30-30
• /
• 2000