Activation of protein kinase Czeta increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated transport

Abstract

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

FIGURE 1.

hOAT3 bait and PKCζ prey constructs. a, schematic of bait/prey constructs relative to native OAT3 and PKCζ proteins. hOAT3-Bait was myc-tagged in the pGBKT7 cloning vector. b, the presence and size of the bait in AH109 haploid yeast was confirmed by Western blot (α-myc antibody). c, following successful mating, replicate plating, and identification of PKCζ as prey by sequencing, bait was confirmed in AH109-Y187 diploid yeast. Myc-tagged p53 positive control was provided by manufacturer.

FIGURE 2.

OAT3 interacts with PKCζ. a, endogenous PKCζ was immunoprecipitated using a specific PKCζ antibody as described under “Experimental Procedures” and rOat3 was detected in the Western blot using a specific rOat3 antibody (left panel). As a control, precipitation of PKCζ itself was confirmed in the stripped blot (right panel). b, a reciprocal experiment showed that following immunoprecipitation with the OAT3, PKCζ was detected (left panel). Once again as control, the presence of rOat3 was also verified in the stripped blot (right panel). Abbreviations: L, total protein lysate; PC, precleared cell lysate; Neg, negative control, i.e. background binding of proteins to beads without antibody; IB, immunoblot.

FIGURE 3.

Effect of insulin on ES transport and PKCζ activity in renal cortical slices. a, insulin and PKCζ, slices from rat or mouse kidney were preincubated for 30 min in buffer alone (Control), or buffer containing 2 μm PKCζ pseudosubstrate inhibitor (PKCζ-PS), 15 (mouse) or 30 (rat) μg/ml insulin, or insulin plus PKCζ-PS. Following preincubation, the slices were incubated for 30 min with 0.1 μm [³H]ES. Data are expressed as tissue to medium ratios, i.e. tissue content (DPM/mg) ÷ medium (DPM/μl). b, PKCζ activity, rat kidney slices were incubated as in a, and the activity of PKCζ was determined as described under “Experimental Procedures.” c, comparison of pseudosubstrate inhibitors, the effect of PKCζ-PS, PKCη-PS, and PKCθ-PS on insulin stimulation of ES uptake by rat cortical slices. All conditions were identical to those described for a. In each graph, data are expressed as mean values for experiments conducted in at least 3 different animals ± S.E. Differences significant at the p < 0.05 level are denoted by asterisks.

FIGURE 4.

Specificity of insulin stimulation of OAT-mediated transport. a, ES, the transport of the OAT3-specific substrate, 1 μm [³H]ES, was determined as described in the legend to Fig. 3a using cortical slices from wild-type and OAT3-null mice. b, PAH, transport of 10 μm [³H]PAH, a substrate for both OATs 1 and 3, was measured in slices from wild-type and OAT3-null mice. c, adefovir, the effects of insulin on transport of the OAT1-specific substrate [³H]ADF (0.1 μm in rat and 1 μm in mouse) were determined in rat and mouse kidney slices. Note: mouse Oat1 does not transport adefovir (11). Data are expressed as tissue to medium ratios (T/M). Each experiment was conducted in slices from three animals. Asterisks indicate significant differences from control values; p < 0.05.

FIGURE 5.

Effect of EGF on transport of estrone sulfate in wild-type and OAT3-null mice. Renal cortical slices were incubated as described in the legend to Fig. 3a with 50 ng/ml EGF or EGF plus 2 μm PKCζ-PS, followed by incubation with 0.1 μm [³H]ES for 30 min. Data are shown as tissue to medium ratios (T/M). Asterisks indicate significant difference from controls, p < 0.05.

FIGURE 6.

Impact of PKCζ inhibition on PKA-mediated stimulation of ES transport by rat renal cortical slices. Rat renal cortical slices were preincubated in buffer containing 75 μm Bt₂cAMP for 20 min (this Bt₂cAMP concentration was shown in preliminary experiments to double uptake of 0.1 μm [³H]ES), followed by a 30-min incubation with and without inhibitors. a, inhibition of EGF-mediated stimulation of ES uptake by H89 and PKCζ-PS. Addition of 2 μm PKCζ-PS, 100 μm H-89, or both blocked EGF stimulation of ES uptake. b, inhibition of insulin-mediated stimulation of ES uptake by H89 and PKCζ-PS. Inhibitors additions were as in a. Data are shown as tissue to medium ratios. Asterisks indicate significant differences from controls, p < 0.05.

FIGURE 7.

Effect of microtubule and microfilament disruptors on insulin stimulation of ES uptake. Rat renal cortical slices were preincubated with buffer containing 2 μm PKCζ-PS, 50 μg/ml brefeldin A (BFA), 50 μm nocodazole, or 50 mg/ml cytochalasin D in the absence or presence of 30 μg/ml insulin. Uptake of 0.1 μm [³H]ES was then measured and calculated as described in the legend to Fig. 3a. Data are shown as tissue to medium ratios. Asterisks indicate significant differences from controls, p < 0.05.