Cytosolic Aryl Sulfotransferase 4A1 Interacts with the Peptidyl Prolyl Cis-Trans Isomerase Pin1

Abstract

Sulfonation by cytosolic sulfotransferases plays an important role in the metabolism of both endogenous and exogenous compounds. Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase found primarily in neurons in the brain. It is highly conserved between species, but no substantial enzyme activity has been identified for the protein. Consequently, little is known about the role of this enzyme in the brain. We performed a yeast two-hybrid screen of a human brain library to isolate potential SULT4A1-interacting proteins that might identify the role or regulation of the sulfotransferase in humans. The screen isolated the peptidyl-prolyl cis-trans isomerase Pin1. Its interaction with SULT4A1 was confirmed by coimmunoprecipitation studies in HeLa cells and by in vitro pull-down of expressed proteins. Moreover, Pin1 binding was dependent on phosphorylation of the SULT4A1 protein. Pin1 destabilized SULT4A1, decreasing its half-life from more than 8 h to approximately 4.5 h. This effect was dependent on the isomerase activity of Pin1 and was inhibited by okadaic acid, suggesting a role for the phosphatase PP2A. Pin1-mediated SULT4A1 degradation did not involve the proteosomes or macroautophagy, but it was inhibited by the calpain antagonists N-acetyl-Leu-Leu-Nle-CHO and Z-Val-Phe-CHO. Finally, Pin1 binding was mapped to two threonine-proline motifs (Thr 8 and Thr 11 ) that are not present in any of the other human cytosolic sulfotransferases. Our findings suggest that SULT4A1 is subject to post-translational modification that alters its stability in the cell. These modifications may also be important for enzyme activity, which explains why specific substrates for SULT4A1 have not yet been identified.

Expression of the Orphan Cytosolic Sulfotransferase SULT4A1 and Its Major Splice Variant in Human Tissues and Cells: Dimerization, Degradation and Polyubiquitination

“…The generation of FLAG-tagged and HA-tagged human wild-type SULT4A1 plasmids (pFLAG-SULT4A1-WT and pHM6-SULT4A1-WT, respectively) has been described previously [13]. To generate the FLAG-tagged human variant SULT4A1 plasmid (pFLAG-SULT4A1-VAR), the same primers and procedure were used as in Mitchell et al [13] except that the template for the PCR was cDNA from IMR-32 cells, which only express the variant SULT4A1 transcript.…”

“…To generate the FLAG-tagged human variant SULT4A1 plasmid (pFLAG-SULT4A1-VAR), the same primers and procedure were used as in Mitchell et al [13] except that the template for the PCR was cDNA from IMR-32 cells, which only express the variant SULT4A1 transcript. The pFLAG-SULT4A1-TV>AE and pFLAG-SULT4A1-KTV>QAE mutants were generated by the GENEART site-directed mutagenesis system (Life Technologies) using pFLAG-SULT4A1-WT as template and the following forward and reverse primers; FP-TV>AE, 5′-GGAAGGACA-TCTTC G CCG AG TCCATGAATGAGAAG-3′, RP-TV>AE 5′-CTTCTCATTCATGG-A CT CGG C GAAGATGTCCTTCC-3′, FP-KTV>QAE 5′-AGAGTTGGGCTGTGG G A-GGACATCTTC-3′, RP-KTV>QAE 5′-GAAGATGTCCT C CCACAGCCCAACTCT-3′, respectively.…”

Regulation of Mouse Brain-Selective Sulfotransferase Sult4a1 by cAMP Response Element-Binding Protein and Activating Transcription Factor-2

“…Crystallography studies suggest that the active site for SULT4A1 is smaller than that of other sulfotransferases and that it may not be able to accommodate the cofactor 3Ј-phosphoadenosine-5Ј-phosphosulfate (Allali-Hassani et al, 2007). We have shown that SULT4A1 is a binding partner for the peptidyl-prolyl cis-trans isomerase Pin1 and that Pin1 modulates the stability of SULT4A1 in a calpaindependent manner (Mitchell and Minchin, 2009). Pin1 selectively binds to phosphoserine/threonine-proline motifs in the trans conformation (Verdecia et al, 2000).…”

Activity Suppression Behavior Phenotype in SULT4A1 Frameshift Mutant Zebrafish

“…Recent work has described the post-translational modification of SULT4A1 via phosphorylation/dephosphorylation as well as a possible interaction with the peptidyl-prolyl cis-trans isomerase, Pin1 (Mitchell and Minchin, 2009;Mitchell et al, 2011). Yet little is known about the exact biologic function that SULT4A1 plays on a molecular level, and understanding its role in the regulation of activity will require extensive work.…”

“…This is possibly due to the absence of a key tryptophan residue present in the catalytically active SULTs that is thought to pi-stack with the adenosine ring of PAPS (Falany et al, 2000;Allali-Hassani et al, 2007). Previous work has suggested that SULT4A1's lack of activity in vitro could also be due to the protein's post-translational modification in vivo (Mitchell and Minchin, 2009;Mitchell et al, 2011).…”