Regulation Of Proline-Directed Phosphorylation Site, Threonine 53 On Dopamine Transporter

The dopamine transporter (DAT) is a plasma membrane protein that clears extraneuronal dopamine (DA) and thus controls the spatio-temporal dynamics of dopaminergic neurotransmission. Also, DAT is the major target for psychostimulant substrates, amphetamine (AMPH) and methamphetamine (METH), and psychostimulant uptake blocker, cocaine (COC). DAT is a phosphoprotein with both the N- and C-termini facing toward the cytosol, with multiple phosphorylation sites on the N-terminus. DAT has a closely spaced 6-serine cluster on the distal N-terminus that is phosphorylated in a protein kinase C (PKC)-dependent manner and a recently identified proline-directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases ERK, JNK and p38. Current studies indicate that COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although the mechanism of drug action on DAT is not completely known, phosphorylation conditions of DAT have been found to be associated with altered surface expression and activity of DAT. In this study, we examined the effect of several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho-specific antibody against phosphorylated T53 (pT53) on DAT. A detailed analysis of pT53 on DAT was performed in LLC-PK1 cells expressing rat DAT (rDAT), rat striatal synaptosomes and in vivo in male Sprague-Dawley rats. Our studies revealed psychostimulant substrates but not uptake blockers significantly stimulated pT53. Pretreatment with COC blocked the AMPH stimulation of pT53 indicating that AMPH stimulates pT53 in a DAT-dependent manner. In rat striatal synaptosomes, METH-stimulation of pT53 was very fast; occurring within 60 sec. Subcutaneous injections of METH in rats stimulated pT53 in a time-dependent manner. Our study demonstrates that the proline-directed phosphorylation site, pT53 is subject to differential regulation by psychostimulant drugs. Prolyl cis-trans isomerase, Pin1 catalyzes the cis-trans isomerization of pThr-Pro peptides allowing the dephosphorylation by conformation-specific phosphatases. We investigated the regulation of pT53 by Pin1 using Juglone (Jug), a small molecule inhibitor of Pin1. Treatment with Jug in LLC-PK1 rDAT cells and rat striatal synaptosomes revealed significant stimulation of pT53. Jug treatment enhances [3H]DA efflux from rat striatal synaptosomes. ELISA indicated interaction between the N-terminus of DAT and Pin1. This is the first evidence of DAT regulation by Pin1. Our study demonstrates the regulation of DAT pT53 by Pin1 and psychostimulant drugs under physiological conditions

Ephrin B Activate Src Family Kinases in Fibroblasts Inducing Stromal Remodeling in Prostate Cancer

Through stromal-epithelial interactions, carcinoma associated fibroblasts (CAF) play a critical role in tumor growth and progression. Activation of erythrophoyetin-producing human hepatocellular (Eph) receptors has been implicated in cancer. Eph receptor interactions with Ephrin ligands lead to bidirectional signals in the recipient and effector cells. The consequences of continuous reverse Ephrin signaling activation in fibroblasts on prostate cancer (PCa) is unknown. When compared to benign prostate fibroblast, CAF displayed higher expression of Ephrin B1, B2, and B3 ligands (EFNB1, EFNB2, and EFNB3). In this study, we found that continuous activation of EFNB1 and EFNB3 in a benign human prostate stromal cell line (BHPrS1) increased the expression of CAF markers and induced a CAF phenotype. BHPrS1EFNB1 and BHPrS1EFNB3 displayed a pro-tumorigenic secretome with multiple effects on neovascularization, collagen deposition, and cancer cell proliferation, overall increasing tumorigenicity of a premalignant prostate epithelial cell line BPH1 and PCa cell line LNCaP, both in vitro and in vivo. Inhibition of Src family kinases (SFK) in BHPrS1EFNB1 and BHPrS1EFNB3 suppressed EFNB-induced ɑ-SMA (Alpha-smooth muscle actin) and TN-C (Tenascin-C) in vitro. Our study suggests that acquisition of CAF characteristics via SFK activation in response to increased EFNB ligands could promote carcinogenesis via modulation of TME in PCa