Rac1 Inhibits Apoptosis in Human Lymphoma Cells by Stimulating Bad Phosphorylation on Ser-75

Laboratory of Biochemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892-4555, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 08/2004; 24(14):6205-14. DOI: 10.1128/MCB.24.14.6205-6214.2004
Source: PubMed


The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are
not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2
family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation
leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro.
Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor
representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that
endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt
is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis
in response to cancer chemotherapy drugs.

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Available from: Baolin Zhang, Dec 27, 2013
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    • "There are several reports of Pak1 stimulating BAD phosphorylation at both S136 and S112 [15], [16], [23], [24]. While most studies relied on cell based assays, some documented the phosphorylation in vitro, usually using phosphospecific antibodies [16], [24], [36]. We showed here, with radioisotope, a more quantitative assay system, that there was still a substantial phosphorylation of BAD with the S112 and S136 double mutant. "
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    ABSTRACT: Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1. We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2'-Dihydroxy-1,1'-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112. Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding.
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    • "The GTPase Rac1 is a key molecule for transformation driven by oncogenic Ras through the cytoskeletal reorganization [18]. The activation of Rac1 by Tiam-1 was described to be associated with tumor progression in T cell lymphoma [19], and Rac1 was also reported to inhibit apoptosis in the human lymphoma cells through the activation of Bad phosphorylation [20]. Unlike what was determined for oncogene "
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    ABSTRACT: DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.
    Preview · Article · Mar 2010 · Biochemical and Biophysical Research Communications
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    • "Rac1-induced activation of its downstream effector p21-activated kinase 1 (PAK1) leads to phosphorylation of the anti-apoptotic protein Bad on serine S75. This protects human lymphoma cells from drug-induced caspase activation and subsequent apoptosis [59]. While overexpression of RhoH suppresses Rac1 activity thus leading to increased apoptosis (see following paragraph) [27], low expression levels of RhoH might increase Rac1-induced protection from apoptosis, drug resistance and cause bad prognosis. "
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    ABSTRACT: Rho GTPases are a distinct subfamily of the superfamily of Ras GTPases. The best-characterised members are RhoA, Rac and Cdc42 that regulate many diverse actions such as actin cytoskeleton reorganisation, adhesion, motility as well as cell proliferation, differentiation and gene transcription. Among the 20 members of that family, only Rac2 and RhoH show an expression restricted to the haematopoietic lineage. RhoH was first discovered in 1995 as a fusion transcript with the transcriptional repressor LAZ3/BCL6. It was therefore initially named translation three four (TTF) but later on renamed RhoH due to its close relationship to the Ras/Rho family of GTPases. Since then, RhoH has been implicated in human cancer as the gene is subject to somatic hypermutation and by the detection of RHOH as a translocation partner for LAZ3/BCL6 or other genes in human lymphomas. Underexpression of RhoH is found in hairy cell leukaemia and acute myeloid leukaemia. Some of the amino acids that are crucial for GTPase activity are mutated in RhoH so that the protein is a GTPase-deficient, so-called atypical Rho GTPase. Therefore other mechanisms of regulating RhoH activity have been described. These include regulation at the mRNA level and tyrosine phosphorylation of the protein's unique ITAM-like motif. The C-terminal CaaX box of RhoH is mainly a target for farnesyl-transferase but can also be modified by geranylgeranyl-transferase. Isoprenylation of RhoH and changes in subcellular localisation may be an additional factor to fine-tune signalling. Little is currently known about its signalling, regulation or interaction partners. Recent studies have shown that RhoH negatively influences the proliferation and homing of murine haematopoietic progenitor cells, presumably by acting as an antagonist for Rac1. In leukocytes, RhoH is needed to keep the cells in a resting, non-adhesive state, but the exact mechanism has yet to be elucidated. RhoH has also been implicated as a regulatory molecule in the NFκB, PI3 kinase and Map kinase pathways. The recent generation of RhoH knockout mice showed a defect in thymocyte selection and TCR signalling of thymic and peripheral T-cells. However, RhoH-deficient mice did not develop lymphomas or showed obvious defects in haematopoiesis.
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