Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.

Orr, George A.; Han, E. K.-H.; Browne, Paul C.; Nieves, Edward; O'Connor, Brigid M.; Yang, Chia-Ping Huang; Horwitz, Susan Band

doi:10.1016/s0021-9258(19)74570-0

Cited by 65 publications

(15 citation statements)

References 47 publications

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“…The internucleotide linker region of PGPcontains several consensus PKC phosphorylation sites (Chen et al, 1986). Ser671 is the site identified in human PGP that is phosphorylated by PKC in vitro (Chambers et al, 1992), and is equivalent to Ser669 identified as the major phosphorylation site in murine mdr 1 b (Orr et al, 1993). Our results indicate that Ser671 appears to be crucial for both drug-stimulated and PKC-stimulated ATPase activity since mutation of Ser671 completely negated the stimulatory effect of PKCa and partially inhibited the effect of verapamil.…”

Section: Discussionmentioning

confidence: 99%

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

1994

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The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.

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Section: Discussionmentioning

confidence: 99%

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

1994

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“…The 17 aa replacement is comprised of mainly glycine and serine residues organized in a Gly 4 Ser configuration which is predicted to be flexible (Figure 1B). This partial linker region deletion, Pgp-(∆653-686), spans amino acids 653-686 and also includes the in vitro and in vivo serine phosphorylation sites of Pgp (21,(38)(39)(40). NIH3T3 cells were transfected with the retroviral vectors pHa-neo (41), pHaMDR1(wild-type), pHaMDR1-(∆653-686), and pHaMDR1-(17 aa replacement) and selected in 30 and 50 ng/mL vincristine.…”

Section: Insertion Of a Flexible Peptide Into The Linker Region Ofmentioning

confidence: 99%

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport

et al. 1998

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P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.

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“…A predominant and tough obstacle in cancer treatment is the acquisition of drug resistance, especially multidrug resistance (MDR). Studies of relevant mechanisms have proved P-glycoprotein (P-gp) as a major mediator [151][152][153][154]. Importantly, many CCBs of all subclasses (phenylalkylamine, dihydropyridine, and benzothiazepine type) and other calcium antagonists are capable to inhibit the P-gp-mediated drug efflux and represent modulators of MDR [155].…”

Section: Blockers Of Vgccsmentioning

confidence: 99%

Calcium signaling in cancer progression and therapy

Lian

Zhao

2021

The FEBS Journal

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VGCCs voltage-gated calcium channels SOCE store-operated calcium entry CRAC calcium release-activated Ca 2+ TRP Transient receptor potential ER endoplasmic reticulum SR sarcoplasmic reticulum IP3 inositol 1,4,5-trisphosphate GPCRs G-protein-coupled receptors MCU mitochondrial calcium uniporter CaMKII Ca 2+ -calmodulin-dependent protein kinase II CACNA1E calcium voltage-gated channel subunit alpha1 E CCBs calcium channel blockers

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Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.

Cited by 65 publications

References 47 publications

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport

Calcium signaling in cancer progression and therapy

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