Tammy Flagg

Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions

Bcl2 functions to suppress apoptosis and retard cell cycle entry. Single-site phosphorylation at serine 70 (S70) is required for Bcl2s antiapoptotic function, and multisite phosphorylation at threonine 69 (T69), S70, and S87 has been reported to inactivate Bcl2. To address this apparent conflict and identify the regulatory role for Bcl2 phosphorylation in cell death and cell cycle control, a series of serine/threonine (S/T) → glutamate/alanine (E/A) mutants including T69E/A, S70E/A, S87E/A, T69E/S70A/S87A (EAA), T69A/S70E/S87A (AEA), T69A/S70A/S87E (AAE), T69E/S70E/S87E (EEE), and T69A/S70A/S87A (AAA) was created to mimic or abrogate, respectively, either single-site or multisite phosphorylation. The survival and cell cycle status of cells expressing the phosphomimetic or nonphosphorylatable Bcl2 mutants were compared. Surprisingly, all of the E but not the A Bcl2 mutants potently enhance cell survival after stress and retard G1/S cell cycle transition. The EEE Bcl2 mutant is the most potent, indicating a possible cumulative advantage for multisite phosphorylation of Bcl2 in survival and retardation of G1/S transition functions. Because the E-containing Bcl2 mutants, but not the A-containing mutants, can more potently block cytochrome c release from mitochondria during apoptotic stress, even at times when steady-state expression levels are similar for all mutants, we conclude that phosphorylation at one or multiple sites within the flexible loop domain of Bcl2 not only stimulates antiapoptotic activity but also can regulate cell cycle entry.

Bcl2's Flexible Loop Domain Regulates p53 Binding and Survival

ABSTRACT p53 not only functions as a transcription factor but also has a direct, apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53-Bcl2 binding is associated with decreased Bcl2-Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2s flexible loop regulatory domain (FLD), since purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (amino acids [aa] 32 to 68). Deletion of the negative regulatory region (Δ32-68) abolishes Bcl2-p53 binding and enhances Bcl2s antiapoptotic function. Conversely, removal of a positive regulatory region (aa 69 to 87) of the FLD, which contains the Bcl2 phosphorylation site(s) T69, S70, and S87, enhances Bcl2-p53 binding and significantly abrogates Bcl2s survival activity. The phospho-mimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and potently inhibits p53-induced cytochrome c release from isolated mitochondria. Furthermore, the FLD-only aa32-87 and aa32-68 peptides but not the aa69-87 peptide can directly bind p53 in vitro. p53-induced cytochrome c release occurs through a mechanism involving Baxs integral insertion into the outer mitochondrial membrane. Either DNA damage to cells or expression of p53 selectively targeted to the mitochondria results in Bcl2-p53 binding followed by exposure of Bcl2s BH3 domain in association with inactivation of Bcl2s antiapoptotic function, indicating a conformational change in Bcl2 can occur upon direct ligation of p53. Thus, Bcl2s FLD contains both positive and negative regulatory regions which functionally regulate Bcl2s antiapoptotic activity by affecting Bax or p53 binding.

Bcl2 negatively regulates DNA double-strand-break repair through a nonhomologous end-joining pathway.

Bcl2 can enhance susceptibility to carcinogenesis, but the mechanism(s) remains fragmentary. Here we discovered that Bcl2 suppresses DNA double-strand-break (DSB) repair and V(D)J recombination by downregulating Ku DNA binding activity, which is associated with increased genetic instability. Exposure of cells to ionizing radiation enhances Bcl2 expression in the nucleus, which interacts with both Ku70 and Ku86 via its BH1 and BH4 domains. Removal of the BH1 or BH4 domain abrogates the inhibitory effect of Bcl2 on Ku DNA binding, DNA-PK, and DNA end-joining activities, which results in the failure of Bcl2 to block DSB repair as well as V(D)J recombination. Intriguingly, Bcl2 directly disrupts the Ku/DNA-PKcs complex in vivo and in vitro. Thus, Bcl2 suppression of the general DSB repair and V(D)J recombination may occur in a mechanism by inhibiting the nonhomologous end-joining pathway, which may lead to an accumulation of DNA damage and genetic instability.

Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

Protein kinase Cζ (PKCζ) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKCζ in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKCζ in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCζ activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCζ specific inhibitor (a myristoylated PKCζ pseudosubstrate peptide). Purified, active PKCζ directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCζ mutant results in Bax phosphorylation at serine 184. PKCζ co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCζ by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCζ mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCζ can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCζ may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.

Bcl2 suppresses DNA repair by enhancing c-Myc transcriptional activity.

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex.

Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSα is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSα activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.

Functional characterization of the murine Tnk1 promoter

Tnk1/Kos1 is a non-receptor protein tyrosine kinase found to be a tumor suppressor. It negatively regulates cell growth by indirectly suppressing Ras activity. We identified and characterized the critical cis-elements required for Tnk1/Kos1s promoter activity. Results indicate that the murine Tnk1 promoter lacks a conventional TATA, CAAT or initiator element (Inr) but contains multiple transcription start sites. Transcription is initiated by a TATA-like element composed of an AT rich sequence at -30 (30 bp upstream) from the major transcription start site and an Inr-like element that overlaps the multiple start sites. Deletion analysis of the m-Tnk1 promoter reveals the presence of both positive (-25 to -151) and negative (-151 to -1201) regulatory regions. The three GC boxes which bind Sp1 and Sp3 with high affinity, an AP2 site (that overlaps with an AML1 site) and a MED1 site comprise the necessary cis-elements of the proximal promoter required for both constitutive and inducible Tnk1/Kos1 expression. Importantly, results reveal that cellular stress reverses the repression of Tnk1/Kos1 and induces its expression through increased high affinity interactions between nuclear proteins Sp1, Sp3, AP2 and MED1 for the m-Tnk1 promoter. These findings provide a mechanism by which the m-Tnk1 promoter can be dynamically regulated during normal growth.

Abstract 4181: Absence and/or functional loss of the tumor suppressor Tnk1 is associated with various human neoplasms

The protein tyrosine kinase activity of Tnk1 (Thirty-eight negative kinase) is required for cell growth suppression due to the fact that the kinase-dead Tnk1 triggers aberrant cell growth, including anchorage independent growth with hyperactivation of Ras (Hoare et. al. 2003; Azoitie et.al. 2007). Mechanistically, the cell growth suppression is associated with the inhibition of Ras activity and the down-stream Raf1-MAPK growth pathway (Hoare et.al. 2003) resulting in G2-M phase cell cycle arrest. A tumor suppressor role of Tnk1 was subsequently confirmed by developing the Tnk1 knockout mouse model by homologous recombination. The TNK1 knockout mice develop many types of tumors upon aging, with B-cell lymphomas, hepatomas, hepatocellular carcinomas, lung adenomas and adenocarcinomas being the most frequent malignancies (Hoare et.al. 2008; May et. al. 2010). Significantly, 40% of Tnk1-/-mice and 26% of Tnk1+/− mice had more than one tumor type develop, likely reflecting Tnk19s broad tumor suppressor function. To confirm the tumor suppressor function of the 72 kDa Tnk1 in human tumors and cell lines, we screened for Tnk19s expression and analyzed its intrinsic/auto kinase activity and in vitro kinase activity. Results indicate that in tumors and cell lines, Tnk1 is either expressed or absent. In B-/T-lymphoma and lung adenocarcinoma cell lines, to name a few, such as Ramos, Jurkat, REH and A549, absence of Tnk1 is associated with aberrant Ras activity, suggesting that loss of Tnk1 expression may contribute to tumor development. In patient specimens and cell lines namely, K562, T47D, MCF7, NCI H82, NCI H387, HEPG2 and HUH, the expressed Tnk1 is found to lack auto-kinase activity and is therefore, inactive in vivo. This data indicates that tumor growth depends on the maintenance of Tnk1 in its inactive form through interaction(s) with scaffolding protein(s) and/or due to SNPs found in its kinase domain. Strikingly, the Hodgkin lymphoma cell line L540 is found to express a tyrosine phosphorylated 60 kDa truncated Tnk-(C17ORF61) fusion gene product (termed tTnk1) raising doubt that it is an activated form of Tnk1 (Gu et. al. 2010). Interestingly, we found that t-Tnk1 is kinase-dead, as it fails to Y-phosphorylate Grb2, a Tnk1 substrate. This supports the concept that t-Tnk1 and/or functionally inactive Tnk1 expressed in tumor cells may function in a dominant negative manner to maintain tumor growth. In conclusion, Tnk1, in human, also functions as a tumor suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4181. doi:1538-7445.AM2012-4181

Abstract 1350: Transgenic expression of PKR induces a myelodysplastic syndrome/pre-leukemia-like condition in mice

Double stranded (ds) RNA activated protein kinase PKR was discovered over 30 years ago as a downstream mediator of the interferon signal pathway. Initial studies focused on PKR9s anti-virus and pro-inflammatory effects. More recently, PKR has been found to be involved in signal transduction pathways critical for the negative regulation of cell growth and initiation of apoptosis. Significantly, loss of PKR expression/activity has been associated with increased growth of human breast carcinoma, B-cell CLL, and T-cell ALL using patient samples. Furthermore, PKR activity is increased in bone marrow progenitor cells isolated from patients with myelodysplastic syndrome (MDS) which undergo significant apoptosis compared to normal marrow progenitors not observed when evolved to AML. Thus, in the process of MDS evolution to leukemia, PKR expression/activity may be downregulated by a process that is currently unclear. To explore the role of PKR in MDS/AML we have constructed a transgenic mouse model expressing human PKR (TgPKR) under control of vav regulatory elements to drive expression specifically in hematopoietic-tissue. We hypothesized transgenic expression of human PKR would exert an inhibitory effect on mouse hematopoiesis, cause ineffective blood cell production, and thus simulate a disease process similar to human MDS. In our preliminary data, quantitative PCR and immunoblotting results confirm that human PKR is specifically expressed in spleen, thymus and bone marrow (BM) but not in liver, kidney, intestine, muscle and other tissues outside hematopoiesis in TgPKR mice. Aberrant activation of transcription factor STAT1 and MAPKinases ERK1/2 occurs in hematopoietic cells from the TgPKR mice. In addition, inhibitory cytokines including interferon gamma, CXCL9 and 10 are overexpressed. Initial characterization of BM cells from TgPKR mice indicates a higher level of uninduced/basal apoptosis in growth medium and increased apoptosis upon withdrawal of growth factors compared to cells from wild type (WT) littermates. Furthermore, BM cells from TgPKR mice display >20% reduced colonies (CFU-GM and CFU-GEMM) which contain 40% less cells than those from WT littermates. Significantly, histopathological analysis of BM and spleen from TgPKR mice reveals a ∼20% increase in bone marrow cellularity with a striking increase in dysplastic megakaryocytes that are either small without nuclear lobation or of normal size but with hypolobation or separated nuclear lobes. Taken together our results demonstrate that transgenic expression of hPKR induces an MDS-like condition in mice characterized by reduced BM cell survival, increased apoptosis and reduced proliferation and differentiation of BM progenitor/stem cells. Thus, increased PKR expression in patients may, at least in part, lead to a BM failure state similar to MDS. Thus, future anti-MDS therapies that inhibit PKR may be clinically useful. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1350. doi:1538-7445.AM2012-1350