c‐Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity‐dependent manner

Abstract

c-Abl is a non-receptor-type tyrosine kinase that regulates various cellular events, including cell proliferation, differentiation, and apoptosis, through phosphorylation of cytoplasmic and nuclear targets. Although we showed that c-Abl induces histone deacetylation, the molecular mechanisms of this phenomenon are largely unknown. Here, we analyzed the effect of c-Abl on the expression of histone deacetylase 1 (HDAC1), because c-Abl was shown to be involved in maintenance of nuclear protein levels of HDAC1. Co-transfection of HDAC1 with c-Abl increased the levels of HDAC1 protein in a kinase activity-dependent manner without affecting its mRNA levels. Treatment with the proteasome inhibitor MG132 increased protein levels of HDAC1 in cells transfected with HDAC1 but not in cells co-transfected with HDAC1 and c-Abl. Among class I HDACs, knockdown of endogenous c-Abl preferentially suppressed endogenous protein levels of HDAC1, suggesting that c-Abl stabilizes HDAC1 protein by inhibiting its proteasomal degradation. Subcellular fractionation showed that the stabilization of HDAC1 by c-Abl occurred in the nucleus. Despite the fact that HDAC1 was phosphorylated by co-expression with c-Abl, stabilization of HDAC1 by c-Abl was not affected by mutations in its sites phosphorylated by c-Abl. Co-expression with HDAC1 and nuclear-targeted c-Abl did not affect HDAC1 stabilization. Therefore, these results suggest that c-Abl induces HDAC1 stabilization possibly through phosphorylation of a cytoplasmic target that is involved in proteasomal degradation of HDAC1.

Src Acts as an Effector for Ku70-dependent Suppression of Apoptosis through Phosphorylation of Ku70 at Tyr-530

“…The mechanism of Src-mediated suppression of apoptosis through Src substrates still remains poorly understood, possibly because protein-tyrosine phosphorylation is extremely unstable within cells. We, therefore, assume that phosphorylation levels of substrates are very rapidly regulated by a balance of the activities between tyrosine kinases and tyrosine phosphatases, like ON/OFF switching in a microprocessor (5,(22)(23)(24)(25)(26)(27)57). Nonetheless, carefully using a high dose of the potent tyrosine phosphatase inhibitor Na 3 VO 4 , we are able to detect tyrosine phosphorylation of endogenous proteins and have shown the significance of tyrosine-phosphorylated substrates (5,24,26,27).…”

“…To construct myc-tagged wildtype Ku70 (myc-Ku70-WT), the BamHI and XhoI sites were created at both ends of human wild-type Ku70 (Ku70-WT; Open Biosystems) by PCR with 5Ј-GAGTCGGATCCTTA-AGCAGTGGAATGTCAGGGTGGGAGTC-3Ј (sense) and 5Ј-GAATAGGGCCCATCTCGAGTCAGTCCTGGAAGT-GCTTGG-3Ј (antisense). Ku70-WT was inserted into the BamHI-XhoI site of the myc-pcDNA3 vector, an myc tag vector (57). The Tyr 3 Phe mutant of Ku70 (myc-Ku70-Y530F) was created by PCR using Ku70-WT as a template with 5Ј-TGGATGAGTTTAAGGAACTAGTTTTCCCACCAGAT-TACAATCCTGAAGG-3Ј (sense) and 5Ј-TGTAATCTG-GTGGGAAAACTAGTTCCTTAAACTCATCCACCAA-GGAGCC-3Ј (antisense).…”

“…Immunoprecipitation was performed using an appropriate primary antibody together with protein-G beads, as described previously (19,21,83,84). To detect tyrosine phosphorylation of Ku70, cell lysates were prepared as described recently (57). In brief, cells were suspended in SDS lysis buffer (0.1% SDS, 50 mM Tris-HCl, pH 7.5, 10 mM Na 3 VO 4 , 10 mM unbuffered HEPES, 4 g/ml aprotinin, 1.6 g/ml pepstatin A, 4 g/ml leupeptin, 1 mM EDTA, and 1 mM PMSF).…”

Effects of BCL2 transfection on the cell cycle and proliferation of human GES-1 cells

“…Cell transfections were conducted according to the Lipofectamine manufacturer instructions (You et al, 2014;Aoyama et al, 2015). Briefly, the steps were as follows: GES-1 cells in the logarithmic growth phase were seeded on 24-well plates, each well containing 0.5 mL RPMI 1640 medium with 10% FBS; cells were incubated at 37°C in 5% CO 2 until 70% confluence, then the cell culture was discarded and the cells were washed once with serumfree DMEM.…”

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

“…An expression vector for wild-type AKAP8 tagged with an myc epitope at the N terminus (myc-AKAP8-wt) was constructed from cDNA encoding human wild-type AKAP8. cDNA that encode AKAP8 was generated by PCR from HeLa S3 cell cDNAs with 5Ј-CTGACGGTACCGCTGGTTCAATGG-ACCAGGGCTACGGAGGCTACGGG-3Ј (sense) and 5Ј-CTAGCTTCTAGATCATTCTGTGGGAACAGCGTCTTTA-GACTCTGCATC-3Ј (antisense), and the BamHI-XbaI fragment of the PCR product was introduced into the BamHI-XbaI site of the pcDNA3/myc vector as described (34). The Tyr to Phe mutants of AKAP8 were created by PCR using AKAP8-wt as a template with primers as follows: Y51F/Y53F, 5Ј-GTCACCACA-GGCAGTACTTTCAGCTTCGGCCCAGCCTCGTGGGAG-3Ј (sense) and 5Ј-GGCTGGGCCGAAGCTGAAAGTACTGCCT-GTGGTGACACTGGTGTTCTG-3Ј (antisense); Y80F, 5Ј-TGC-CATGCACATGGCTAGCTTCGGCCCAGAGCCATGCACC-GAC-3Ј (sense) and 5Ј-GGCTCTGGGCCGAAGCTAGCCAT-GTGCATGGCAGGGGCCCCGG-3Ј (antisense); Y146F/Y150F/ Y152F/Y154F, 5Ј-CACAACCCCTTCAGGCCTAGCTTCAGC-TTCGACTTTGAGTTCGACCTGG-3Ј (sense) and 5Ј-GTCGA-ACTCAAAGTCGAAGCTGAAGCTAGGCCTGAAGGG-GTTGTGCTCC-3Ј (antisense); Y170F, 5Ј-CAATGGCA-GCTTTGGTGGCCAGTTCAGTGAATGCCGAGACCCAG-CCC-3Ј (sense) and 5Ј-GTCTCGGCATTCACTGAACTG-GCCACCAAAGCTGCCATTGCGGTCGGAC-3Ј (antisense); Y311F, 5Ј-GGAAACGGAAGCAGTTCCAGCTGTTCGAGGA-GCCAGACACCAAACTGG-3Ј (sense) and 5Ј-GGTGTCTGG-CTCCTCGAACAGCTGGAACTGCTTCCGTTTCCTGCCC-GTG-3Ј (antisense); Y436F, 5Ј-GTGGAGTTCCTCCAGGAAT-TCATTGTAAACAGAAATAAGAAAATTG-3Ј (sense) and 5Ј-CTTATTTCTGTTTACAATGAATTCCTGGAGGAACTCC-ACGGTCTTGTC-3Ј (antisense); Y539F, 5Ј-GTGAAGATG-CTCGAGAAATTCCTCAAGGGTGAGGACCCTTTCACC-3Ј (sense) and 5Ј-CACCCTTGAGGAATTTCTCGAGCATCTTC-ACTATATGTCTGTTG-3Ј (antisense).…”