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

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.

A three-dimensional model of human organic anion transporter 1: aromatic amino acids required for substrate transport.

Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site ( approximately 830A(3)). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr(230) (domain 5) and Lys(431) and Phe(438) (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [(3)H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V(max) values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr(230) and Phe(438), which are important for substrate/protein interactions.

Proteasome inhibition alters the transcription of multiple yeast genes.

The 26S proteasome degrades denatured proteins and other proteins targeted for destruction by covalent modification. Here we show that impaired proteasome function influences the transcription of numerous yeast genes. Of 6144 genes present on the macroarray filters used in this study, approximately 5% showed measurable mRNA decreases and 2% showed mRNA increases following 30 min of proteasome inhibition. Northern blot hybridization shows that this response is time- and dose-dependent and occurs with either pharmacological or genetic impairment of the proteasome. A number of splicing factors possess the PEST motif found in certain proteasome substrates. However, mRNA levels drop with proteasome inhibition without obvious increases in intron-bearing pre-mRNA, indicating that splicing is not generally impaired when proteome activity is blocked. Chimeric gene constructs, nuclear run-on experiments, and transcriptional inhibition studies show that for members of three functional groups (i.e., ribosomal protein genes, histone genes, and heat shock protein genes) changes in mRNA levels occur largely by transcriptional modulation. In addition, these studies reveal a possible new means of modulating kinetochore levels through CEP3 expression. Together these data strongly support the view that proteasome activity plays a significant role in the regulation of eukaryotic gene expression.