The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities.

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.

cAMP-dependent cytosolic mislocalization of p27(kip)-cyclin D1 during quinol-thioether-induced tuberous sclerosis renal cell carcinoma.

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.