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Cytosolic Phosphorylation of Calnexin Controls Intracellular Ca2ϩ Oscillations Via an Interaction with Serca2b H

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Cytosolic Phosphorylation of Calnexin Controls Intracellular Ca2ϩ Oscillations Via an Interaction with Serca2b H Cytosolic Phosphorylation of Calnexin Controls Intracellular Ca2ϩ Oscillations via an Interaction with SERCA2b H. Llewelyn Roderick,* James D. Lechleiter,‡ and Patricia Camacho* *Department of Physiology, and ‡Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900 Abstract. Calreticulin (CRT) and calnexin (CLNX) are of this residue. We also demonstrate by coimmunopre- lectin chaperones that participate in protein folding in cipitation that CLNX physically interacts with the the endoplasmic reticulum (ER). CRT is a soluble ER COOH terminus of SERCA2b and that after dephos- lumenal protein, whereas CLNX is a transmembrane phorylation treatment, this interaction is significantly protein with a cytosolic domain that contains two con- reduced. Together, our results suggest that CRT is sensus motifs for protein kinase (PK) C/proline- uniquely regulated by ER lumenal conditions, whereas directed kinase (PDK) phosphorylation. Using confo- CLNX is, in addition, regulated by the phosphorylation cal Ca2ϩ imaging in Xenopus oocytes, we report here status of its cytosolic domain. The S562 residue in that coexpression of CLNX with sarco endoplasmic CLNX acts as a molecular switch that regulates the reticulum calcium ATPase (SERCA) 2b results in inhi- interaction of the chaperone with SERCA2b, thereby bition of intracellular Ca2ϩ oscillations, suggesting a affecting Ca2ϩ signaling and controlling Ca2ϩ-sensitive functional inhibition of the pump. By site-directed mu- chaperone functions in the ER. tagenesis, we demonstrate that this interaction is regu- lated by a COOH-terminal serine residue (S562) in Key words: phosphorylation • calnexin • ER lectin CLNX. Furthermore, inositol 1,4,5-trisphosphate– chaperones • Ca2ϩ ATPases • Ca2ϩ signaling mediated Ca2ϩ release results in a dephosphorylation Introduction 2ϩ Multiple cellular processes, including the control of gene IP3-induced Ca oscillations in Xenopus oocytes (Ca- transcription (Dolmetsch et al., 1998; Li et al., 1998), growth macho and Lechleiter, 1993). We have demonstrated pre- cone formation in neurons (Gomez and Spitzer, 1999), and viously that calreticulin (CRT), a lectin chaperone located activation of mitochondrial respiration (Robb-Gaspers et in the ER, inhibits these oscillations and specifically tar- al., 1998) are encoded by spatial–temporal aspects of intra- gets SERCA2b (Camacho and Lechleiter, 1995a; John et cellular Ca2ϩ oscillations. Repetitive cytosolic Ca2ϩ tran- al., 1998). The COOH terminus of SERCA2b, unlike that sients in Xenopus oocytes are under the control of two ma- of SERCA2a, faces the ER lumen and contains an as- jor opposing factors: Ca2ϩ release through the inositol paragine residue that forms part of a consensus site for 1 1,4,5-trisphosphate receptor channel (IP3R) (Iino, 1990; N-linked glycosylation (N1036) (Gunteski-Hamblin et al., Parker and Ivorra, 1990; Bezprozvanny et al., 1991; Finch 1988) that we demonstrated is required for interaction et al., 1991) and Ca2ϩ reuptake into the ER Ca2ϩ store by with CRT (John et al., 1998). From these data we con- the Ca2ϩ ATPases (Camacho and Lechleiter, 1993; MacLen- cluded that a functional interaction between CRT and nan et al., 1997). Sarco endoplasmic reticulum calcium SERCA2b may be required for inhibition of these oscilla- ATPase (SERCA)-type ATPases control the frequency of tions. CRT is a soluble lumenal ER protein, whereas calnexin (CLNX) and its testis-specific isoform, calmegin (CLMG), Address correspondence to Patricia Camacho, Department of Physiology, are type I transmembrane (TM) proteins (Michalak et al., University of Texas Health Science Center at San Antonio, 7703 Floyd 1992; Bergeron et al., 1994; Ohsako et al., 1994; Watanabe Curl Drive, San Antonio, TX 78229-3900. Tel.: (210) 567-6558. Fax: (210) et al., 1994). This family of molecular chaperones is char- 567-4410. E-mail: [email protected] 1 acterized by a lumenal domain that contains a motif of 17 Abbreviations used in this paper: CLMG, calmegin; CLNX, calnexin; amino acids repeated three times in CRT and four in CRT, calreticulin; IP3R, inositol 1,4,5-trisphosphate receptor; PDK, pro- line-directed kinase; PK, protein kinase; SERCA, sarco endoplasmic CLNX and CLMG (Fliegel et al., 1989; Michalak et al., reticulum calcium ATPase; TM, transmembrane. 1992; Ohsako et al., 1994; Watanabe et al., 1994). This do- The Rockefeller University Press, 0021-9525/2000/06/1235/13 $5.00 The Journal of Cell Biology, Volume 149, Number 6, June 12, 2000 1235–1247 http://www.jcb.org 1235 main is responsible for interaction of the chaperone with primer with sequence 5Ј-CCTTGTGATCCTCTTCTGCTGTGCTGGA- the monoglucosylated form of N-linked glycoproteins dur- AAGAAACAGTCC-3Ј and reverse primer with sequence 5Ј-GGA- CTGTTTCTTTCCAGCACAGCAGAAGAGGATCACAAGG-3Ј, and ing protein folding in the ER (Vassilakos et al., 1998). The using as template pHNb-CLNX. The CLNX-S562A mutant was generated presence of this conserved domain led us to test whether using the forward primer with sequence 5Ј-GGATGAAATTTTGAA- CLNX, like CRT, also inhibits Ca2ϩ oscillations. Here, we CAGAGCACCAAGAAACAGAAAGCCACGAAG- 3Ј, and the com- demonstrate that CLNX inhibits Ca2ϩ oscillations in a man- plementary reverse primer with sequence 5Ј-CTTCGTGGCTTTCT- ner consistent with inhibition of SERCA2b. Furthermore, GTTTCTTGGTGCTCTGTTCAAAATTTCATCC-3Ј and pHNb-CLNX 2ϩ was again used as template. The double mutant CLNX-S485A/S562A was as was the case for CRT, repetitive Ca waves were unaf- generated by exchanging the RsaI to NheI fragment containing the fected by coexpression of CLNX with a SERCA2b mutant CLNXS562A mutation for the equivalent wild-type cDNA in the vector lacking the lumenal asparagine (SERCA2b-N1036A). pHNb-CLNXS485A backbone. The presence of both mutations was con- In contrast to CRT, CLNX has in its cytosolic domain firmed by sequencing. The CLNX cytosolic peptide was generated by con- ventional PCR amplification of an internal fragment spanning the S562 consensus sites for phosphorylation by protein kinase PKC consensus site (A537 to the COOH terminus). This primer had se- (PK) C, casein kinase II, PKA, and proline-directed kinase quence 5Ј-ACTGGGATCCATGGATGCTGAAGAAGATGGTGG-3Ј (PDK) (Tjoelker et al., 1994; Wong et al., 1998). Recently, and incorporated a BamHI site at its 5Ј end. The antisense oligonucleotide serine 562 located close to the COOH terminus of CLNX had sequence 5Ј-GCTTAGAGACTCCATTC-3Ј, which is complemen- has been shown to support phosphorylation by the PDK, tary to the 3Ј untranslated region of Xenopus ␤-globin. The product of this PCR amplification was digested with BamHI and XbaI and after puri- extracellular-signal regulated kinase 1 (ERK-1) (Wong et fication was subcloned into vector pHNb and similarly digested (plasmid al., 1998), and was implicated in regulating an interaction pHNb-CLNXcyt). The expression vector for SERCA2b has been de- of CLNX with ribosomal proteins (Chevet et al., 1999). scribed elsewhere (Camacho and Lechleiter, 1995a; John et al., 1998). The second residue, S485, faces the cytosol in proximity to The COOH-terminal domain mutants of SERCA2b and SERCA2b- N1036A spanning TM segments 9–11 were generated by PCR using as for- the single TM segment. However, phosphorylation of this ward primer 5Ј-ACTGGGATCCATGCCCCCCTGGGAGAACATCT- residue has eluded detection (Wong et al., 1998). Since ac- GGCTCG-3Ј incorporating a BamHI site and as the reverse primer tivation of the IP3 signaling pathway results in the produc- 5Ј-GCTTAGAGACTCCATTC-3Ј, which is complementary to the 3Ј untranslated region of Xenopus ␤-globin present in the template (pHN- tion of both IP3 and diacylglycerol (DAG), which is a known activator of PKC, we have also tested whether the SERCA2b). The PCR products were digested with BamHI and HindIII and ligated to the similarly digested Xenopus expression vector, pHN. The two consensus motifs for PKC/PDK phosphorylation in final constructs encode the COOH terminus of SERCA2b and ϩ CLNX play a role in the modulation of Ca2 oscillations. SERCA2b-N1036A starting at the methionine residue 924, which is lo- Here we demonstrate that when the S562 is mutated to an cated just before TM9 (Bayle et al., 1995). The plasmids were named unreactive alanine (S562A), Ca2ϩ oscillations are no longer pHN-SERCA2b/TM9-11 and pHN-SERCA2b-N1036A/TM9-11. A simi- lar strategy was followed to generate the SERCA 2a/TM9-10 using as inhibited, suggesting that S562 in CLNX plays a critical PCR template plasmid pHN-SERCA2a (John et al., 1998). All vectors 2ϩ role in the regulation of Ca oscillations. The presence of generated were fully sequenced automatically by the core facility at the the S485 was required for regulation by S562. By immuno- UTHSCSA. precipitation of CLNX from 32P-labeled oocytes, we dem- onstrate that CLNX becomes de-phosphorylated in S562 In Vitro Transcriptions and Oocyte Protocols 2ϩ after mobilization of intracellular Ca by IP3. Further- Synthetic mRNA was prepared as described previously (Camacho and more, we demonstrate by coimmunoprecipitation that Lechleiter, 1995a). Plasmids were linearized with NotI, except for plasmid CLNX physically interacts with the COOH terminus of pHNb-CLNX, which was linearized by NheI digestion. Transcription initi- ated at the T7 promoter was performed using the Megascript™ high yield SERCA2b and that this interaction is reduced when aspar- transcription kit and capped with m7G(5Ј)ppp(5ЈЈ) (both from Ambion). agine 1036 is mutated
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