Inactivation of glycogen synthase kinase 3␤ (GSK3␤)is critical for transcription of atrial natriuretic factor (ANF) by ␤-adrenergic receptors in cardiac myocytes. We examined the mechanism by which GSK3␤ regulates ANF transcription. Stimulation of ␤-adrenergic receptors induced nuclear accumulation of GATA4, whereas ␤-adrenergic ANF transcription was suppressed by dominant negative GATA4, suggesting that GATA4 plays an important role in ␤-adrenergic ANF transcription. Interestingly, GATA4-mediated transcription was markedly attenuated by GSK3␤. GSK3␤ physically associates with GATA4 and phosphorylates GATA4 in vitro. Overexpression of GSK3␤ suppressed both basal and ␤-adrenergic increases in nuclear expression of GATA4, whereas inhibition of GSK3␤ by LiCl caused nuclear accumulation of GATA4, suggesting that GSK3␤ negatively regulates nuclear expression of GATA4. The nuclear exportin Crm1 reduced nuclear expression of GATA4, and the reduction was enhanced by GSK3␤ but not by kinase-inactive GSK3␤. Leptomycin B, an inhibitor for Crm1, increased basal nuclear GATA4 and suppressed GSK3␤-induced decreases in nuclear GATA4. These results suggest that GSK3␤ negatively regulates nuclear expression of GATA4 by stimulating Crm1-dependent nuclear export. Inhibition of GSK3␤ by ␤-adrenergic stimulation abrogates GSK3␤-induced nuclear export of GATA4, causing nuclear accumulation of GATA4, which may represent an important signaling mechanism mediating cardiac hypertrophy.

Angiotensin II (Ang II) type 1 receptors (AT1Rs) activate tyrosine kinases, including Src. Whether or not tyrosine kinase activation by AT1R occurs independently of heterotrimeric G protein coupling and, if so, the cellular function of such a mechanism are unknown. To address these questions, we used an AT1aR intracellular second loop mutant, which lacks heterotrimeric G protein coupling (AT1a-i2m). Surprisingly, Ang II-induced Src activation was preserved in AT1a-i2m, which was not attenuated by inhibiting protein kinase C and Ca 2؉or by inhibiting G␣ i or G␣ q in CHO-K1 cells. By contrast, Ang II-induced Src activation was abolished in a C-terminally truncated AT1a-(1-309), where Ang II-induced inositol phosphate response was preserved. Ang II activates ERKs via a Src-Ras-dependent mechanism in AT1a-i2m. ERKs activated by AT1a-i2m phosphorylate their cytoplasmic targets, including p90 RSK , but fail to translocate into the nucleus or to cause cell proliferation. Ang II-induced nuclear translocation of ERKs by wild type AT1aR was inhibited by overexpression of nuclear exportin Crm-1, while that by AT1a-i2m was restored by leptomycin B, an inhibitor of Crm-1. In summary, while Src and ERKs are activated by Ang II even without heterotrimeric G protein coupling, the carboxyl terminus of the AT1 receptor is required for activation of Src. Interestingly, ERKs activated by heterotrimeric G protein-independent mechanisms fail to phosphorylate nuclear targets due to lack of inhibition of Crm-1-induced nuclear export of ERKs. These results suggest that heterotrimeric G protein-dependent and -independent signaling mechanisms play distinct roles in Ang II-mediated cellular responses.The signaling mechanism of the angiotensin II (Ang II) 1 type 1 (AT1) receptor has traditionally been portrayed as being dependent on heterotrimeric G proteins (1). The AT1 receptor activates phospholipase C␤ (PLC␤) via G␣ q proteins. This causes generation of inositol trisphosphates as well as diacylglycerol, which in turn causes release of Ca 2ϩ from the intracellular Ca 2ϩ store sites and activation of protein kinase C (PKC), respectively. The AT1 receptor also couples to G␣ i , thereby regulating adenylyl cyclase (2). Besides coupling with the heterotrimeric G proteins, activation of tyrosine kinases is also intimately involved in the AT1 receptor signaling (3, 4). Both nonreceptor type tyrosine kinases (Src, Fyn, Yes, Pyk2, focal adhesion kinase, and JAK2) and receptor type tyrosine kinases (EGF and platelet-derived growth factor receptors) are activated by the AT1 receptor (5-8). These tyrosine kinases regulate downstream signaling mechanisms, including PLC␥, Ras-Raf-MEK-ERK, and STAT (6, 9, 10), thereby playing a critical role in cell growth responses by Ang II.Several mechanisms have been shown to mediate tyrosine kinase activation by heterotrimeric G protein-coupled receptors (GPCRs; reviewed in Refs. 11-13). First, the downstream effectors of G␣ and G␤␥ mediate tyrosine kinase activation. For example, Ca 2ϩ and PKC activated through ...

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Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.The signaling mechanism of the angiotensin II (Ang II) 1 type 1 (AT1) receptor has traditionally been portrayed to be dependent on heterotrimeric G proteins, including G␣ q and G␣ i proteins and their downstream targets, primarily phospholipase C (1). This results in inositol triphosphate generation, which in turn causes an increase in intracellular calcium concentrations and diacylglycerol formation, leading to activation of protein kinase C. However, recent investigations revealed that tyrosine phosphorylation is also intimately involved in AT1 receptor signaling (2-6). Ang II-induced ERK1/2 activation, for example, requires tyrosine kinase activation, including Src family tyrosine kinases (7, 8) and epidermal growth factor receptor (EGFR) (9, 10). It is unclear, however, how AT1 receptors, which lack intrinsic tyrosine kinase activities, are able to stimulate tyrosine kinases.We have recently shown that an AT1 receptor second intracellular loop mutant, lacking heterotrimeric G protein coupling, is able to activate Src tyrosine kinase (11). This suggests that heterotrimeric G protein-independent mechanisms are able to activate Src. Furthermore, increasing lines of evidence suggest that the carboxyl terminus (C-tail) of the AT1 receptor ...

Chaga mushrooms have been used in folk and botanical medicine as a remedy for cancer, gastritis, ulcers, and tuberculosis of the bones. A 72-year-old Japanese female had been diagnosed with liver cancer 1 year prior to presenting at our department. She underwent hepatectomy of the left lobe 3 months later. Chaga mushroom powder (4 - 5 teaspoons per day) had been ingested for the past 6 months for liver cancer. Renal function decreased and hemodialysis was initiated. Renal biopsy specimens showed diffuse tubular atrophy and interstitial fibrosis. Oxalate crystals were detected in the tubular lumina and urinary sediment and oxalate nephropathy was diagnosed. Chaga mushrooms contain extremely high oxalate concentrations. This is the first report of a case of oxalate nephropathy associated with ingestion of Chaga mushrooms.