Potent inhibition of human sulfotransferase 1A1 by 17α-ethinylestradiol: role of 3'-phosphoadenosine 5'-phosphosulfate binding and structural rearrangements in regulating inhibition and activity.

Sulfotransferase (SULT) 1A1 is the major drug/xenobiotic-conjugating SULT isoform in human liver because of its broad substrate reactivity and high expression level. SULT1A1 sulfates estrogens with low micromolar K(m) values consistent with its affinity for sulfation of many small phenolic compounds. Binding studies showed the unexpected ability of 17α-ethinylestradiol (EE2) to bind and inhibit SULT1A1 activity toward p-nitrophenol and ß-naphthol at low nanomolar concentrations, whereas EE2 was not sulfated until significantly higher concentrations were reached. EE2 had a K(i) of 10 nM for inhibiting p-nitrophenol and ß-naphthol sulfation and inhibited 17ß-estradiol (E2) sulfation in intact human MCF-7 breast cancer cells with a K(i) of 19 nM. In contrast, the K(m) for EE2 sulfation by SULT1A1 was 700 nM. The K(d) for EE2 binding of pure SULT1A1 was 0.5 ± 0.15 µM; however, the K(d) for EE2 binding to the SULT1A1-PAP complex was >100-fold lower (4.3 ± 1.7 nM). The K(d) for E2 binding to SULT1A1 changed from 2.3 ± 0.9 to 1.2 ± 0.56 µM in the presence of PAP. Docking studies with E2 indicate that E2 binds in a competent orientation in the resolved structure of SULT1A1 in the both presence and absence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). However, EE2 binds in a catalytically competent orientation in the absence of PAPS but in a noncompetent orientation via formation of a charge interaction with Tyr108 if PAPS is bound first. In conclusion, EE2 is a potent inhibitor, but not a substrate, of SULT1A1 at low nanomolar concentrations, indicating the possibility of drug-drug interactions during contraceptive therapy.

With-No-Lysine Kinase 3 (WNK3) stimulates glioma invasion by regulating cell volume.

Among the most prevalent and deadly primary brain tumors, high-grade gliomas evade complete surgical resection by diffuse invasion into surrounding brain parenchyma. Navigating through tight extracellular spaces requires invading glioma cells to alter their shape and volume. Cell volume changes are achieved through transmembrane transport of osmolytes along with obligated water. The sodium-potassium-chloride cotransporter isoform-1 (NKCC1) plays a pivotal role in this process, and previous work has demonstrated that NKCC1 inhibition compromises glioma invasion in vitro and in vivo by interfering with the required cell volume changes. In this study, we show that NKCC1 activity in gliomas requires the With-No-Lysine Kinase-3 (WNK3) kinase. Western blots of patient biopsies and patient-derived cell lines shows prominent expression of Ste-20-related, proline-alanine-rich kinase (SPAK), oxidative stress response kinase (OSR1), and WNK family members 1, 3, and 4. Of these, only WNK3 colocalized and coimmunoprecipitated with NKCC1 upon changes in cell volume. Stable knockdown of WNK3 using specific short hairpin RNA constructs completely abolished NKCC1 activity, as measured by the loss of bumetanide-sensitive cell volume regulation. Consequently, WNK3 knockdown cells showed a reduced ability to invade across Transwell barriers and lacked bumetanide-sensitive migration. This data indicates that WNK3 is an essential regulator of NKCC1 and that WNK3 activates NKCC1-mediated ion transport necessary for cell volume changes associated with cell invasion.