Molecular Immunology. Vol. 32, No. 3, pp. 167-175, 1995 zyxwvutsrqpo Elsevier Science Ltd 0161-5890(94)00142-1 Printed in Great Britain RAG-l AND RAG-2 GENE EXPRESSION AND V(D)J RECOMBINASE ACTIVITY ARE ENHANCED BY PROTEIN PHOSPHATASE 1 AND 2A INHIBITION IN LYMPHOCYTE CELL LINES ADRIAN M. CASILLAS,* ANDREW D. THOMPSON,? SAMUEL CHESHIER,* SANTIAGO HERNANDEZ* and RENATO J. AGUILERA*$ *Department of Biology, Division of Cell, Molecular and Developmental Biology; and tMolecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90024, U.S.A. (First received 24 May 1994; accepted in revised form 23 September 1994) Abstract-Expression of the recombination activating genes, RAG-l and RAG-2, in lymphocytes, has been shown to depend on second messenger systems. An increase in intracellular CAMP upon stimulation with caffeine increases RAG expression while activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) results in decreased RAG expression. The stringent regulation of recombination appears to be partially dependent on protein kinase activities which, alone, are not likely to be sufficient to regulate recombinase activity. We provide evidence implicating a role for serine/threonine phosphatases in the signal transduction pathway which regulates RAG gene expression and consequently the recombination process in lymphocytes. The cell permeable tumor promoter, calyculin-A (CLA), which is a potent inhibitor of the type 1 and 2A serine/threonine protein phosphatases (PPI and PP2A, respectively), was shown to upregulate the expression of RAG-l and RAG-2 in pre-B as well as mature B- and T-lymphocyte cell lines. Although agents such as caffeine known to increase intracellular CAMP levels induce RAG expression, synergy between CLA and caffeine was not detected in pre-B cells. An in vivo assessment of recombination activity after transfection of pre-B cells with an extrachromosomal recombination vector revealed a moderate increase in recombinase activity which paralleled RAG expression after CLA stimulation. Although increased CAMP levels in pre-B cells has been associated with upregulation of RAG expression we found no such upregulation in a surface immunoglobulin M positive zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM (sIgM+ ) cell line, WEHI-231, and a T cell receptor positive (TCR+ ) murine cell line, EL-4. Moreover, in these mature lymphocyte cell lines there was no evidence of synergy in the regulation of RAG-l and RAG-2 mRNA upon stimulation with CLA and caffeine. These results suggest novel intracellular mechanisms for the upregulation of RAG gene expression and confirm a role for type 1 and 2A phosphatases in the control of RAG gene expression and recombinase activity in lymphocyte cell lines. Key words: recombinase activating genes, V(D)J recombination, calyculin-A, protein phosphatase inhibitor. INTRODUCTION The regulation of V(D)J recombination in lymphocytes by second messengers has recently been explored through the transcriptional regulation of the recombi- nase activating genes RAG-l and RAG-2 (Menetski and Gellert, 1990; Turka et al., 1991; Ma et al., 1992). The expression of these genes is necessary and sufficient to allow recombination in pre-B and pre-T cells as well as fibroblast cell lines transfected with RAG-l and RAG-2 SAuthor to whom correspondence should be addressed at: UCLA Department of Biology, Los Angeles, CA 90024, U.S.A. Abbreviations: CAMP, cyclic adenosine monophosphate; CLA, calyculin-A; DP, double positive; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol myristate acetate; PPI, protein phosphatase I; PPZA, protein phosphatase 2A; RAG-l, recombinase activating gene-l; RAG-2, re- combinase activating gene-2; SP, single positive. (Oettinger et al., 1990). Intracellular second messengers such as CAMP and PKA have been shown to rapidly increase gene expression of CAMP-responsive genes followed by a gradual attenuation due to downregula- tion (Sasaki et al., 1984). In pre-B cell lines, agents which increase intracellular CAMP levels upregulate RAG-l and RAG-2 expression with a concomitant increase in recombinase activity (Menetski and Gellert, 1990). Moreover, RAG gene expression is not apparently down- regulated directly upon expression of surface Ig (Ma et al., 1992). Rather, crosslinking of this receptor in immature sIg+ B cells appears to activate transcriptional downregulation or attenuation of the RAG-l and RAG-2 genes (Ma et al., 1992). The mechanisms of RAG downregulation appears to require PKC activation, as PMA and ionomycin treatment have been shown to rapidly downregulate V(D)J recombinase activity and RAG expression in immature lymphocytes (Menetski and Gellert, 1990; Turka et al., 1991). Furthermore, 167