Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 3710-3714, May 1991 Biochemistry Calcium/calmodulin-dependent protein kinase mediates a pathway for transcriptional regulation (Rous sarcoma virus/long terminal repeat/CAAT/enhancer binding protein/prolactin gene) MICHAEL S. KAPILOFF*t, J. MICHAEL MATHIS*, CHARLES A. NELSON*t, CHIJEN R. LIN*, AND MICHAEL G. ROSENFELD*t tHoward Hughes Medical Institute, and *Eukaryotic Regulatory Biology Program, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0648 Communicated by Morris E. Friedkin, January 17, 1991 ABSTRACT Calcium influx in response to extracellular prepare soluble extracts (17). Binding of antibody and cal- signals can modulate gene transcription. A constitutive, calci- modulin to nitrocellulose blots of SDS/PAGE size- um/calmodulin-independent mutant of type H calcium/ fractionated whole-cell protein was performed as described calmodulin-dependent protein kinase was capable ofincreasing (17). CaMKa activity in bacterial soluble extracts was as- the transcription rate ofspecific genes independently ofprotein sayed by using microtubule-associated protein 2 (MAP-2) as kinase C activation. This increase was mediated by transfer- a substrate (18). MAP-2 was separated from other radiola- nable cis-active elements capable of binding the transcription beled proteins by SDS/PAGE. factor CAAT/enhancer binding protein. Therefore, the acti- Transfectional Analyses. RSV CaMKa mutants were con- vation oftype II calcium/calmodulin-dependent protein kinase structed by substituting the appropriate sequence into a in response to stimuli that increase intracellular calcium is RSV-neor expression vector (19, 20). Rat prolactin [-3 proposed to represent a distinct second messenger pathway in kilobase pairs (kbp) or -422 bp to +36 bp] and growth calcium-mediated regulation of gene transcription. hormone (-316 to +7 bp) promoter reporter plasmids were described (21, 22). The mouse primase p49 promoter reporter Second messengers such as cAMP and calcium ions can plasmid was the generous gift of B. Tseng and C. Prussak activate serine/threonine protein kinases that modulate gene (University ofCalifornia San Diego, La Jolla, CA). One copy transcription (1, 2). In contrast to cAMP, calcium seems of the cAMP response element (CRE) from the a-chorionic capable of stimulating multiple signaling pathways (3, 4), gonadotropin gene (-208 to -185 bp) (1) or two copies of a including protein kinase C (5, 6). Brain-specific type II phorbol ester response element (PRE) (23) were inserted into calcium/calmodulin-dependent protein kinase (CaM-kinase) a -36- to +34-bp rat prolactin reporter plasmid (22). is a member of a family of evolutionarily conserved, widely An RSV LTR luciferase reporter plasmid was constructed expressed isozymes (7) implicated in exocytosis, long-term for expression studies. After conversion to Xho I of the Nde potentiation, and induction of oocyte maturation (8-10). I site of RSV-chloramphenicol acetyltransferase (24), the Because calmodulin antagonists can affect the rate of tran- Xho I to HindIII fragment (RSV LTR included) was sub- scription of the prolactin, fos, and proopiomelanocortin cloned into pBluescript. After conversion to Not I ofthe Aat genes, a calmodulin-dependent protein kinase may regulate II site in a pSV2ALA5'luciferase derivative (25), the com- gene transcription (5, 6, 11-13). To investigate a role for plete luciferase cDNA and simian virus 40 (SV40) termination CaM-kinase in transcriptional regulation, constitutively ac- sequences were transferred to pBluescript RSV LTR as a tive CaM-kinase mutants were engineered to isolate the HindIII/Not I fragment. Sequencing showed that the LTR potential effects of CaM-kinase from other calcium- fragment was of the nearly identical Schmidt-Ruppin strain dependent events. Calcium/calmodulin-independent forms A. The deletion series of the RSV LTR was designed as of the brain CaM-kinase a subunit (CaMKa) specifically follows: a unique Bgl II restriction site was introduced at activated the rat prolactin promoter and the Rous sarcoma -59, -111, -169, -285, and -488 bp with subsequent virus long terminal repeat (RSV LTR) promoter in cotrans- transfer of the appropriate Xho I/Bgl II and Bgl II/HindIII fection assays with cultured GC rat pituitary tumor cells. A fragments to form the complete set of10 deletion mutants. All CaM-kinase response element (CaMRE) is described that is deletions affected baseline activity, in agreement with pub- capable ofselectively conferring induction oftranscription by lished data (26). Double-stranded oligonucleotides (see Fig. activated CaM-kinase. 3C) corresponding to RSV strain D were inserted into the -36- to +34-bp prolactin promoter (22) and/or the Bgl II site of the -169-bp RSV LTR reporter plasmids. METHODS GC cells were transfected with DEAE-dextran as de- Bacterial Expression of CaMKa. The complete cDNA for scribed by using 10 ug ofplasmid DNA per 3 x 106 cells (20). CaMKa was constructed by fusing overlapping C3 and C31 After transfection, cells were incubated for 2 days in Dul- clones encoding partial rat sequences (14). After introduction becco's modified Eagle's medium containing 10%o fetal bo- of a Nde I site overlapping the codon for the initiator vine serum. Cells were induced with dibutyryl cAMP (1 mM methionine by site-directed mutagenesis, the bacterial final concentration) or phorbol 12-myristate 13-acetate expression vectors for CaMKa were constructed by insertion ofthe coding sequences into plasmid pT7-7 (15). pT7-CaMKa Abbreviations: CaM-kinase, type II calcium/calmodulin-dependent was expressed as described (16), and induced cells were protein kinase; CaMKa, CaM-kinase brain a subunit; C/EBP, either lysed into SDS/PAGE sample buffer or were used to CAAT/enhancer binding protein; RSV LTR, Rous sarcoma virus long terminal repeat; CaMRE, CaM-kinase response element; PMA, phorbol 12-myristate 13-acetate; MAP-2, microtubule-associated The publication costs of this article were defrayed in part by page charge protein 2; CRE, cAMP response element; PRE, phorbol ester payment. This article must therefore be hereby marked "advertisement" response element; SV40, simian virus 40. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3710 Downloaded by guest on September 27, 2021 Biochemistry: Kapiloff et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3711 (PMA; 1 ,4M final concentration) when appropriate. Harvest- mutant proteins was assayed with the substrate MAP-2. As ing and luciferase assays were performed as described (25). shown in Fig. 1C, truncation mutants at residues 326 and 316 DNA Binding Protein Assays. A cDNA encoding CAAT/ displayed wild-type activity, while truncation at residue 290 enhancer binding protein (C/EBP) (27) was isolated from a resulted in constitutive calcium-independent MAP-2 phos- GC library and expressed in bacteria as described above (16). phorylation. Catalytic function was eliminated by the muta- GC nuclear extracts were prepared by the method of Sealey tion oflysine residue 42 to methionine (M42) in the consensus and Chalkley (28), and gel-shift mobility assays were per- ATP binding site (32). formed as described (29). For competition assays, CaMKa 1-290 Specifically Activates Gene Transcription. (NH4)2SO4-fractionated 530 mM nuclear extract (29) was The potential for CaM-kinase to modulate gene transcription bound to 70 pM end-labeled double-stranded oligonucleotide was investigated by cotransfection analyses in GC rat pitu- containing the RSV LTR sequence from -231 to -193 bp, in itary tumor cells (Fig. 2). Expression ofrat prolactin and RSV the presence of the indicated concentration of unlabeled LTR fusion genes was induced 2.7- and 14-fold, respectively, competitor oligonucleotide. DNase I footprinting (29) of the by cotransfection with the constitutive CaMKa 1-290 trun- RSV LTR from -285 to -52 bp was performed with excess cation protein (Fig. 2A). The expression of the rat growth (NH4)2SO4-fractionated 530 mM nuclear extract. hormone and mouse primase promoters was not altered. This induction was dependent on the presence of active CaM- kinase, since transcriptional effects were not observed when RESULTS cells were cotransfected with either unstimulated, wild-type Analysis of CaMKa Mutants. Bacterially expressed mu- enzyme or an inactivated CaMKa 1-290 (lysine to methionine tants of the rat brain CaMKa (Fig. lA) truncated prior to or at residue 42) mutant [1.1 ± 0.1- and 1.2 + 0.3-fold induction following the putative calmodulin-binding domain (14) were of RSV LTR, respectively (n = 3)]. RSV LTR inductions characterized by using a specific antibody or biotinylated were also obtained in cotransfections by using two full-length calmodulin (Fig. 1B). All mutant proteins migrated at the mutant forms of CaMKa containing constitutive activity expected molecular weight; an additional band at Mr 32,000 (M.S.K., unpublished data). (lanes WT and M42), resulting from a bacteria-specific trun- Reporter plasmids containing either CREs (1) or PREs (23) cation (31), was not detected with in vitro translation prod- were not regulated by the addition of constitutive CaMKa. ucts (F. Cruzalegui and M.S.K., unpublished data). Of the These results suggest that CaMKa does not regulate specific four mutations assayed, only truncation at residue 290 abol- gene induction via activation of protein kinase A or protein ished calmodulin binding (Fig. 1B, lane 13). Calcium- kinase C, even though bacterially expressed CRE binding dependent phosphorylation activity of the wild-type and protein is an effective in vitro CaM-kinase substrate (M.S.K., unpublished observations). Consistent with distinct func- A. Ca+l/CaM Dependent Protein Kinase tions of protein kinase C and CaM-kinase, addition of the Type 11 Brain cx-subunit phorbol ester PMA to cells cotransfected with CaMKa 1-290 activated the RSV LTR reporter plasmid approximately c CM Ca A. Z Luaferase SV40 ATP DFG SPE CaM cDNA splice polyA Fold-Induction by CaMKa 1-290 Binding Site Box Box Binding 5 10 15 Protein Kinase Regulatory Association (-3 kbp Domain Domain Domain Prolactin -422 bp Hormone B.