Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2.
Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.
Stolk
Meaghan
M
Department of Physiology & Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Cooper
Elizabeth
E
Vilk
Greg
G
Litchfield
David W
DW
Hammond
James R
JR
D015762:2-Chloroadenosine / Q000378:metabolism; D001859:Bone Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D047390:Casein Kinase II / Q000172:deficiency / Q000502:physiology*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D003199:Computer Systems; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics / Q000502:physiology*; D033722:Equilibrative-Nucleoside Transporter 2 / Q000235:genetics / Q000502:physiology*; D005573:Formycins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D009705:Nucleosides / Q000378:metabolism; D012516:Osteosarcoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism
D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D005573:Formycins; D009705:Nucleosides; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; D014316:Tritium; C034561:formycin B; D015762:2-Chloroadenosine; D013868:Thioinosine; D047390:Casein Kinase II; C001789:4-nitrobenzylthioinosine