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T C Chambers

Publications and source records attributed to T C Chambers.

At least 19 recordsLinked to original sources
PubMed2001

AP-1 activation and altered AP-1 composition in association with increased phosphorylation and expression of specific Jun and Fos family proteins induced by vinblastine in KB-3 cells.

Vinblastine and other microtubule inhibitors are important antitumor agents that cause mitotic arrest, and induce apoptosis through poorly understood mechanisms, in a wide variety of cell lines. The activating protein 1 (AP-1) transcription factor is a major target of the c-Jun NH2-terminal kinase (JNK) signaling pathway, which is activated by microtubule inhibitors. Therefore, we examined the effect of vinblastine on AP-1 composition and activity in human KB-3 carcinoma cells. Vinblastine caused highly selective effects on AP-1 proteins in a concentration- and time-dependent manner. Specifically, c-Jun, expressed at a low level in control cells, was greatly increased and phosphorylated, Jun D was phosphorylated, Jun B underwent phosphorylation and subsequently became undetectable, and Fra 1 expression was also greatly increased. In contrast. Fra 2, c-Fos, and Fos B were relatively unchanged by vinblastine. Changes in AP-1 preceded caspase 3 activation and, therefore, occurred prior to the commitment phase of apoptosis. With the exception of c-Jun, which was not affected by paclitaxel, the same alterations in AP-1 proteins occurred after exposure to vincristine, paclitaxel, and colchicine, demonstrating that these are general responses to microtubule inhibition. Supershift assays demonstrated that in control cells, AP-1 binding activity was mediated by Jun D/Fra 2 heterodimers, whereas after vinblastine treatment, AP-1 complexes also containing c-Jun and Fra 1 were present, suggesting that induction of these latter proteins by vinblastine is functionally significant. Consistent with these observations, vinblastine stimulated AP-1-dependent luciferase reporter gene transcription. These findings suggest that alterations in AP-1 composition and activity may be key events in the early response of KB-3 cells to microtubule inhibitors.

Antineoplastic Agents, Phytogenic↗
PubMed2001

The c-Jun NH(2)-terminal protein kinase/AP-1 pathway is required for efficient apoptosis induced by vinblastine.

Vinblastine is an important antitumor agent that induces G(2)-M arrest and subsequent apoptosis in a wide variety of cell lines, but the molecular mechanisms that link mitotic arrest and apoptosis are poorly understood. The AP-1 transcription factor has been implicated in many critical cellular processes, including apoptosis, and is a major target of the c-Jun NH(2)-terminal kinase signaling pathway that is activated by vinblastine and other microtubule inhibitors. In this study we sought to determine the role of c-Jun NH(2)-terminal kinase/AP-1 in the response of KB3 carcinoma cells to vinblastine. For this purpose, we generated KB3 cell lines that stably expressed the c-Jun dominant-negative deletional mutant TAM67, which lacks the NH(2)-terminal transactivation domain. KB3-TAM67 cell lines displayed normal growth kinetics and essentially unaltered basal AP-1 activity, but vinblastine-induced phosphorylation of c-Jun and activating transcription factor-2, and AP-1 activation, were strongly inhibited. KB3-TAM67 cell lines arrested normally at G(2)-M in response to vinblastine, but were significantly more resistant to the drug, exhibiting markedly delayed apoptosis and increased overall survival, relative to control cells. To investigate the underlying mechanisms, differential expression of apoptotic regulatory genes was monitored by immunoblot and cDNA microarray analysis. We found that vinblastine treatment caused down-regulation of p53 and its target p21 and up-regulation of tumor necrosis factor alpha, Bak, and several other genes in control but not in KB3-TAM67 cells, identifying these genes as putative targets of vinblastine-inducible AP-1. These results demonstrate that vinblastine-inducible AP-1 plays a destructive, proapoptotic role and may do so by regulating the expression of a specific subset of target genes that promotes efficient apoptotic cell death following mitotic arrest.

Activating Transcription Factor 2↗
PubMed2001

Role of mitogen-activated protein kinases in the response of tumor cells to chemotherapy.

Antitumor agents, despite having diverse primary mechanisms of action, mediate their effects by inducing apoptosis in tumor cells. Cellular commitment to apoptosis, or the ability to evade apoptosis in response to damage, involves the integration of a complex network of survival and death pathways. Among the best-characterized pathways regulating cell survival and cell death are those mediated by the mitogen-activated protein kinase (MAPK) family. Not surprisingly, MAPK signaling pathways have been implicated in the response of tumor cells to chemotherapeutic drugs. Indeed, literature in this area has grown enormously in recent years, and the present review attempts to provide an overview and perspective of these advances. While the activities of the major MAPK subgroups are subject to modulation upon exposure of different types of cancer cell lines to diverse classes of antitumor agents, the response tend to be context-dependent, and can differ depending on the system and conditions. Despite these complexities, some important trends have surfaced, and molecular connections between MAPK signaling pathways and the apoptotic regulatory machinery are beginning to emerge. With increased evidence supporting a role for MAPK signaling in antitumor drug action, MAPK modulators may have potential as chemotherapeutic drugs themselves or as chemosensitizing agents. The ability of MAPK/ERK kinase (MEK) inhibitors to block survival signaling in specific contexts and promote drug cytotoxicity represents an example, and recent knowledge of the pro-apoptotic functions of JNK and p38 suggests possible new approaches to targeted therapy. However, it will be important first to extrapolate the knowledge gained from these laboratory findings, and begin to address the role of MAPKs in the clinical response to chemotherapeutic drugs.

Animals↗
PubMed2000

Modulation of mitogen-activated protein kinases and phosphorylation of Bcl-2 by vinblastine represent persistent forms of normal fluctuations at G2-M1.

Microtubule inhibitors, widely used in cancer chemotherapy, induce G2-M arrest and apoptosis and have in common the ability to stimulate Raf-1/Bcl-2 phosphorylation and activate c-Jun NH2-terminal protein kinase (JNK). These signal transduction pathways are thought to be activated in response to microtubule damage to promote apoptosis. However, Bcl-2 phosphorylation has been reported to occur at G2-M in nonapoptotic cells, raising the possibility that this and perhaps other signaling pathways altered by microtubule inhibitors reflect perturbations of normal mitotic events. In this study, we sought to test this hypothesis. We show that Bcl-2 phosphorylation and JNK activation, as well as extracellular response kinase and p38 inactivation, occur not only in response to vinblastine but also as discrete transient events at G2-M phase in untreated synchronized KB-3 cells. Thus, modulation of these pathways is not a response to microtubule damage; rather they occur normally at G2-M, and it is the extent, duration, and/or irreversible nature of the signals that distinguish a preapoptotic cell from one destined to divide. These findings provide novel insight into the relationship between mitotic and apoptotic signaling and the mechanism of action of antimitotic drugs.

Activating Transcription Factor 2↗
PubMed2000

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade.

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells.

Microtubule inhibitors are widely used in cancer chemotherapy, but the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. Because members of the mitogen-activated protein kinase (MAPK) family have been implicated in regulation of cell survival and cell death, we examined the extent and kinetics of activation of JNK, ERK, and p38 MAPKs in response to treatment of KB-3 carcinoma cells with several microtubule inhibitors. All four agents tested (vinblastine, vincristine, Taxol, and colchicine) caused significant (6- to 13-fold) activation of JNK, concomitant inactivation of ERK, and a reduction in basal p38 MAPK activity. JNK activation and ERK inactivation occurred prior to caspase 3 activation. The microtubule inhibitors also induced phosphorylation of Raf-1 kinase. SEK-1, upstream of JNK, was also activated and phosphorylated in response to the microtubule inhibitors, and sustained phosphorylation of three endogenous JNK substrates (c-Jun, ATF-2, and JunD) was observed. By comparison, the antitumor agent doxorubicin induced activation of JNK and p38 but had no effect on ERK activity or Raf-1. These data demonstrate that microtubule inhibitors elicit distinct and specific effects on MAPK-mediated signaling pathways and suggest in particular that coordinate and reciprocal alterations in JNK and ERK activities are important facets of the cellular response to microtubule disruption.

Caspase 3↗
PubMed2000

Two-drug combinations that increase apoptosis and modulate bak and bcl-X(L) expression in human colon tumor cell lines transduced with herpes simplex virus thymidine kinase.

Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5- to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X(L), was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X(L). In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X(L) protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.

Alkaloids↗
PubMed1999

Differential phosphorylation of sites in the linker region of P-glycoprotein by protein kinase C isozymes alpha, betaI, betaII, gamma, delta, epsilon, eta, and zeta.

To determine whether individual protein kinase C (PKC) isozymes differentially phosphorylate sites in the linker region of human P-glycoprotein (P-gp), we used a synthetic peptide substrate, PG-2, exactly corresponding to amino acid residues spanning the region 656-689 of the multidrug resistance gene (MDRI). All tested PKC isozymes phosphorylated PG-2. The maximum phosphate incorporation by calcium-dependent PKC isozymes alpha, betaI, betaII, and gamma was 3, 2, 2, and 3 mol phosphate/mol PG-2, respectively. The maximum phosphate incorporation by calcium-independent isozymes delta, epsilon, eta, and zeta was 1.5, 0.5, 1.5, and 1.5 mol phosphate/mol PG-2, respectively. Two-dimensional tryptic phosphopeptide mapping indicated differential phosphorylation of the PKC consensus sites Ser-661, Ser-667, and Ser-671 by individual isozymes, which may be functionally significant. These data suggest that differential phosphorylation by PKC isoenzymes of PKC sites within the P-gp linker region may play a role in modulating P-gp activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Phorbol ester induced MDR1 expression in K562 cells occurs independently of mitogen-activated protein kinase signaling pathways.

The MDR1 gene encoding the multidrug pump P-glycoprotein is transcriptionally activated in response to diverse extracellular stimuli, including the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the signal transduction pathway responsible is unknown. Downstream of protein kinase C (PKC), the effects of TPA are often mediated by the Raf-1/MEK/ERK mitogen-activated protein kinase (MAPK) cascade, and Raf-1 has been implicated in MDR1 induction by serum and mitogens. Therefore, we examined the potential role of MAPK activation in TPA-mediated MDR1 induction in human leukemia K562 cells. MDR1 mRNA expression was significantly increased by TPA in the concentration range of 4 - 100 nM, with a maximal response 5 - 10 h after TPA addition. TPA-mediated MDR1 induction was inhibited by several PKC inhibitors including staurosporine, H7 and calphostin C. TPA stimulated the subcellular translocation of PKCalpha from the cytosol to the membrane and nucleus but did not affect other PKC isozymes. TPA also activated the Raf1/MEK/ERK cascade and activated another MAPK member, p38, but not JNK. In order to determine the potential role of MAPKs in MDR1 induction by TPA, specific inhibitors were utilized. The MEK inhibitor PD 098059, as well as the PKC inhibitors, completely blocked TPA-mediated ERK activation. However, under identical conditions, MDR1 induction by TPA was completely unaffected by PD 098059. Furthermore, SB 202190, which effectively inhibited TPA-mediated p38 activation, failed to inhibit TPA-induced MDR1 mRNA expression. These data demonstrate that MDR1 induction by TPA occurs via a PKC-dependent mechanism that operates independently of ERK, p38 or JNK pathways, and thus have important implications for understanding the mechanisms of MDR1 induction by extracellular stimuli.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Differential roles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interleukin-1beta- and tumor necrosis factor-alpha-induced low density lipoprotein receptor expression in HepG2 cells.

The inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF), elevated in inflammatory, malignant, and infectious diseases, induce low density lipoprotein (LDL) receptor transcription in HepG2 cells, and such an induction can account for hypocholesterolemia associated with these states. However, the signaling mechanisms of cytokine-mediated LDL receptor induction are largely unexplored. In the present studies, we examined the potential involvement of different mitogen-activated protein kinase (MAPK) pathways. Northern analysis demonstrated that IL-1beta or TNF significantly increased LDL receptor transcript in HepG2 cells, whereas expression of another tightly regulated sterol-responsive squalene synthase gene was unaffected. IL-1beta treatment resulted in transient activation of three MAPK cascades, namely p46/54(JNK), p38(MAPK), and ERK-1/2, with maximal activation of 20-, 25-, and 3-fold, respectively, occurring 15-30 min after cytokine addition. PD98059, a specific inhibitor of MAPK kinase activity, inhibited IL-1beta-induced LDL receptor expression. In contrast, SB202190, a specific inhibitor of p38(MAPK), enhanced IL-1beta-induced LDL receptor expression, with a concomitant increase in ERK-1/2 activity. Similarly, TNF induced LDL receptor expression also required ERK-1/2 activation. Finally, sterols repressed IL-1beta induced receptor expression, without affecting ERK-1/2 activation. These results show that IL-1beta- or TNF-induced LDL receptor expression requires ERK-1/2 activation, that the p38(MAPK) pathway negatively regulates LDL receptor expression, and that sterols inhibit induction at a point downstream of ERK-1/2 in HepG2 cells.

Animals↗
PubMed1997

Phorbol ester-induced low density lipoprotein receptor gene expression in HepG2 cells involves protein kinase C-mediated p42/44 MAP kinase activation.

The signaling pathway involved in low density lipoprotein (LDL) receptor gene expression induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated in the human hepatoma HepG2 cell line. Treatment of HepG2 cells with 100 nM TPA resulted in an approximately 20-fold increase in LDL receptor mRNA level, as determined by RT-PCR, which peaked at 2-4 h of treatment and subsequently declined. The protein kinase C (PKC) inhibitors calphostin C and staurosporine prevented TPA-mediated LDL receptor mRNA induction. In contrast, TPA did not affect squalene synthase mRNA expression. Immunoblotting of cell extracts with isozyme-specific PKC antibodies revealed that HepG2 cells expressed PKC alpha, which was mainly cytosolic, and PKC beta, PK epsilon, and PKC zeta, all of which were present in both the cytosolic and particulate fractions. Treatment of HepG2 cells with 100 nM TPA resulted in translocation of cytosolic PKC alpha to the particulate fraction, with a maximum at 30 min-2 h of treatment, but was without effect on the subcellular distribution of the other isozymes. TPA treatment also led to activation of the mitogen-activated protein kinase (MAPK) ERK cascade. The specific MAPK pathway inhibitor PD98059 blocked TPA-induced ERK activation. Furthermore, pretreatment of cells with PD98059 inhibited TPA-induced LDL receptor mRNA induction. Moreover, pretreatment of cells with calphostin C inhibited TPA-mediated ERK activation and LDL receptor mRNA induction in a dose-dependent fashion. Based on a close kinetic correlation between PKC alpha translocation and ERK activation, and the effects of specific inhibitors, these findings suggest that translocation/activation of PKC alpha, and subsequent activation of the Raf-1/MEK/ERK MAPK cascade, represent key events in the transcriptional induction of LDL receptor gene by TPA in HepG2 cells.

Blotting, Northern↗
PubMed1996

Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs.

c-Jun NH2-terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to many stressful stimuli including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines. In this study, we investigated whether JNK plays a role in the cellular response to different drugs commonly used in cancer chemotherapy. Treatment of human KB-3 carcinoma cells with Adriamycin resulted in a time- and dose-dependent activation of JNK of up to 40-fold. Treatment with vinblastine or etoposide (VP-16) also activated JNK, with maximum increases of 6.5- and 4.3-fold, respectively. Consistent with these findings, increased c-Jun phosphorylation was observed after drug treatment of cells. In contrast, none of the drugs significantly activated the extracellular response kinase/mitogen-activated protein kinase pathway. Since these drugs are transport substrates for the MDR1 gene product, P-glycoprotein, JNK was assayed in two multidrug-resistant (MDR) KB cell lines, KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, basal JNK activity was increased 7-fold in KB-A1 cells and 4-fold in KB-V1 cells, with no change in JNK protein expression, indicating that JNK is present in a more highly activated form in the MDR cell lines. Under conditions optimal for JNK activation, Adriamycin, vinblastine, and VP-16 all induced MDR1 mRNA expression in KB-3 cells. Our findings suggest that JNK activation is an important component of the cellular response to several structurally and functionally distinct anticancer drugs and may also play a role in the MDR phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression, subcellular distribution and response to phorbol esters of protein kinase C (PKC) isozymes in drug-sensitive and multidrug-resistant KB cells evidence for altered regulation of PKC-alpha.

Protein kinase C (PKC) comprises a family of related phospholipid-dependent serine/threonine protein kinases. PKC has been implicated in the induction and maintenance of the multidrug-resistance (MDR) phenotype but the role of different isozymes is not well understood. We compared the expression and subcellular distribution, and membrane association and down-regulation induced by phorbol esters, of individual PKC isozymes in drug-sensitive KB-3 and multidrug-resistant KB-V1 human carcinoma cell lines. Immunoblotting with isozyme-specific antibodies indicated the presence of PKC alpha (cytosol only). PKC beta (membrane only). PKC epsilon (mainly membrane associated) and PKC zeta (both fractions). PKC delta and PKC gamma were not detected. The expression levels of PKC beta. PKC epsilon and PKC zeta were unchanged in KB-V1 cells; PKC alpha was modestly increased ( approximately 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKC alpha were confirmed by immunofluorescent localization studies. Revertant cells, obtained by culturing KB-V1 cells in a drug-free medium, regained drug sensitivity with a loss of P-glycoprotein and a concomitant decrease in expression of PKC alpha, KB-V1 cells were found to differ markedly from KB-3 cells with respect to the translocation and down-regulation specifically of PKC alpha upon exposure to 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA). Treatment with 30 nM TPA for 24 h completely depleted KB-3 cells of PKC alpha whereas 1 microM TPA was required to deplete KB-V1 cells of PKC alpha. Similar results were obtained when phorbol-12, 13-dibutyrate was used instead of TPA. Defective TPA-mediated down-regulation of PKC alpha was also observed in another PKC alpha-overexpressing MDR cell line. KB-A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drug-sensitive and MDR cells. Sensitive and resistant cells exhibited similar expression levels of RACK1, a PKC-binding protein important in activation-induced translocation. These findings further highlight the importance of PKC alpha in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells.

To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glyco-protein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.

3T3 Cells↗
PubMed1996

P-glycoprotein, multidrug resistance and protein kinase C.

The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites.

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.

ATP Binding Cassette Transporter, Subfamily B, Mem↗