Bao Q. Vuong

Pim serine/threonine kinases regulate the stability of Socs-1 protein

Studies of SOCS-1-deficient mice have implicated Socs-1 in the suppression of JAK-STAT (Janus tyrosine kinase-signal transducers and activators of transcription) signaling and T cell development. It has been suggested that the levels of Socs-1 protein may be regulated through the proteasome pathway. Here we show that Socs-1 interacts with members of the Pim family of serine/threonine kinases in thymocytes. Coexpression of the Pim kinases with Socs-1 results in phosphorylation and stabilization of the Socs-1 protein. The protein levels of Socs-1 are significantly reduced in the Pim-1−/−, Pim-2−/− mice as compared with wild-type mice. Similar to Socs-1−/− mice, thymocytes from Pim-1−/−, Pim-2−/− mice showed prolonged Stat6 phosphorylation upon IL-4 stimulation. These data suggest that the Pim kinases may regulate cytokine-induced JAK-STAT signaling through modulation of Socs-1 protein levels.

Specific recruitment of protein kinase A to the immunoglobulin locus regulates class-switch recombination

Immunoglobulin class-switch recombination (CSR) requires activation-induced cytidine deaminase (AID). Deamination of DNA by AID in transcribed switch (S) regions leads to double-stranded breaks in DNA that serve as obligatory CSR intermediates. Here we demonstrate that the catalytic and regulatory subunits of protein kinase A (PKA) were specifically recruited to S regions to promote the localized phosphorylation of AID, which led to binding of replication protein A and subsequent propagation of the CSR cascade. Accordingly, inactivation of PKA resulted in considerable disruption of CSR because of decreased AID phosphorylation and recruitment of replication protein A to S regions. We propose that PKA nucleates the formation of active AID complexes specifically on S regions to generate the high density of DNA lesions required for CSR.

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AIDS38A), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AIDWT). B cells homozygous for the AIDS38A mutation show substantially impaired CSR and SHM, correlating with inability of AIDS38A to interact with endogenous RPA. Moreover, mice haploinsufficient for AIDS38A have even more severely impaired CSR when compared with mice haploinsufficient for AIDWT, with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Protein phosphatase 2A regulates the stability of Pim protein kinases

The pim family of proto-oncogenes encodes three serine-threonine kinases that have been implicated in the development of malignancies in mice and in humans. Expression of the Pim protein kinases is tightly regulated at the transcriptional, post-transcriptional, and translational levels. Dysregulation ofpim transcription and pim mRNA stability have been implicated in Pim-mediated transformation. The data presented herein demonstrate that expression of the Pim kinases is additionally regulated at the post-translational level, by the serine-threonine phosphatase protein phosphatase 2A (PP2A). The catalytic subunit of PP2A associates with the Pim kinases in vivo, and the Pim kinases are substrates of PP2A phosphatase activity in vitro. Furthermore, overexpression of PP2A reduces the levels of the Pim proteins, whereas inhibition of PP2A activity by the protein phosphatase inhibitor okadaic acid stabilizes the Pim proteins. Finally, the effects of PP2A on the expression of the Pim proteins can affect Pim function. Taken together, these data suggest that PP2A activity is important for the regulation of the stability and function of the Pim kinases.

A DNA break- and phosphorylation-dependent positive feedback loop promotes immunoglobulin class-switch recombination

The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.

Combinatorial Mechanisms Regulating AID-Dependent DNA deamination: Interacting Proteins and Post-translational Modifications

Protective humoral immune responses result from immunoglobulin (Ig) diversification reactions that proceed through programmed DNA double-strand breaks and mutations in developing or mature B cells. While primary Ig diversity is dependent on V(D)J recombination and the RAG proteins, secondary diversification is achieved through class switch recombination (CSR) and somatic hypermutation (SHM), which require AID (activation induced deaminase). Because aberrant AID activity can result in mutations in non-Ig loci and DNA translocations between the Ig locus and non-Ig genes, the activity of AID must be stringently regulated. AID mRNA expression is regulated transcriptionally by cytokine stimulation and post-transcriptionally by miRNAs. AID activity is regulated by post-translational modifications, subcellular localization, and interaction with other proteins. All of these molecular mechanisms have evolved to specifically induce AID-dependent mutations and DNA double-strand breaks at the Ig loci to promote maximal Ig gene diversification while limiting the access of this mutator to non-Ig regions.

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70S6k kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.

Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis

ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.

Regulation of Cytokine Signaling

Cytokines are important modulators of the immune response that underlies the inflammatory process in atopic forms of asthma. Interleukin (IL)-4 and IL-13 are important cytokines for the regulation of these asthmatic immune responses. However, the cellular mechanisms that regulate IL-4 and IL-13 signaling remain unknown. Recently, a new family of proteins, termed suppressors of cytokine signaling (SOCS), has been identified. We have previously shown that SOCS-1 is a potent inhibitor of JAK-STAT signaling activated by IL-4. SOCS-1 expression is regulated both at the RNA and protein stability level. To identify proteins that bind and potentially regulate SOCS-1, we used the yeast two-hybrid system. We have identified the serine-threonine kinase Pim-2 as a binding partner for SOCS-1. Our preliminary studies demonstrate that SOCS-1 can interact with all three Pim kinases in mammalian cells. Co-expression of SOCS-1 with Pim kinases leads to the expression of novel SOCS-1 isoforms to require serine-threonine kinase activity. Pim kinases can directly phosphorylate SOCS-1. In addition, co-expression of SOCS-1 with Pim2 increases the levels of SOCS-1 protein. Finally, expression of Pim-2 increases the inhibition of IL-4 signaling by SOCS-1. These data lead to a model by which the expression of Pim kinases alters SOCS-1 function through a phosphorylation event that stabilizes the SOCS-1 protein. This chapter proposes experiments to test this model and determine the role Pim kinases play in regulating IL-4 signaling in vivo. In addition, we propose to study the role of Pim kinases in a murine model of asthma.

CLONING, EXPRESSION AND CRYSTALLIZATION OF PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE FROM BACILLUS STEAROTHERMOPHILUS

Pyrimidine nucleoside phosphorylase (PYNP) from B. stearothermophilus has been cloned and purified for crystallization. Crystals of a potential protein-inhibitor complex have been prepared by co-crystallization techniques using the substrate analog pseudouridine. These crystals provide good-quality diffraction images to 2.7 A and belong to space group P21. The asymmetric unit contains the dimer structure of PYNP with unit-cell parameters a = 53.9, b = 71.9, c = 123.3 A and beta = 96.9 degrees.