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calmodulin-dependent protein kinase I in Xenopus laevis
Comparative Biochemistry and Physiology Part B 134 (2003) 499–507
Calciumycalmodulin-dependent protein kinase I in Xenopus laevis夞 Takeo Saneyoshia,*, Shoen Kumeb, Katsuhiko Mikoshibaa,c,d a Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama, Wako-shi 351-0198, Japan Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan c Department of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan d Calcium Oscillation Project, ICORP, Japan Science and Technology Corporation (JST), 3-14-4 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
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Received 29 July 2002; received in revised form 5 November 2002; accepted 11 December 2002
Abstract Calciumycalmodulin (CaM) dependent protein kinase I (CaM-KI) is a member of a well-defined multi-functional CaM-K family, but its physiological and developmental functions have yet to be determined. Here, we have cloned two cDNAs encoding CaM-KI from a Xenopus laevis (X. laevis) oocyte cDNA library. One is a novel isoform of CaM-KI, named CaM-KI LiKb (XCaM-KI LiKb). The other is an a isoform of CaM-KI (XCaM-KIa), which is a highly related to previously cloned mammalian isoform. XCaM-KIa was constantly expressed through embryogenesis, whereas XCaMKI LiKb expression dramatically increased in the neurula stage. Both XCaM-KI isoforms exhibited kinase activity in a Ca2qyCaM-dependent manner. Overexpression of a constitutively active mutant of CaM-KI isoforms inhibited cell cleavage in X. laevis embryos and caused a marked change of cell morphology in Hela cells. Taken together, these results suggest that CaM-KI plays a role in cell-structure regulation during early embryonic development. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Calcium; Calmodulin; Protein kinase; Xenopus laevis; cDNA cloning; Novel isoform; Cell cleavage arrest
1. Introduction In early embryogenesis, the inositol 1,4,5-trisphosphate (IP3)–Ca2q signal transduction pathway is involved in many biological processes, including fertilization, cell cleavage, and dorsoventral axis formation (Kume et al., 1997; Kume, 1999; Saneyoshi et al., 2002). The participation of 夞 The nucleotide sequences of XCaM-KI LiKb and XCaMKIa have been deposited in DDBJyEMBLyGenBank娃 DNA database under the accession numbers AB082999 and AB083000, respectively. *Corresponding author. Tel.: q81-484679745; fax: q81484679744. E-mail address: [email protected] (T. Saneyoshi).
Ca2q in cell division has been proposed, but its molecular mechanism is not well understood (Muto et al., 1996; Muto and Mikoshiba, 1998; Mitsuyama et al., 1999). The Ca2q ycalmodulin-dependent protein kinase (CaM-K) family has three known related members: CaM-KI, -KII and -KIV (Soderling and Stull, 2001). CaM-KI and IV are closely related protein kinases with many biochemical similarities, and they are regulated by CaM-K kinase (CaM-KK), which is further upstream, through phosphorylation of the Thr residue in the activation loop in vitro, —also a Ca2q ycalmodulin-dependent event. Though CaM-KII and CaM-KIV have been associated with many aspects of cellular functions, the
1096-4959/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959(02)00292-0
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physiological and developmental role of CaM-KI is still unclear (Lorca et al., 1993; Morin et al., ¨ et al., 2000; Means, 2000; Wayman et 1994; Kuhl al., 2000; Soderling et al., 2001). In this paper, we report molecular cloning of two isoforms of Xenopus laevis (X. laevis) CaMKI (XCaM-KI), one is an a isoform of CaM-KI (XCaM-KIa), and the other is a novel isoform of CaM-KI, which named XCaM-KI LiKb. Our data demonstrate that both XCaM-KI isoforms conformed to the properties of the mammalian CaMKI, and the expression of the two XCaM-KI isoform was differentially regulated. Moreover, we show that injection of a constitutively active XCaM-KI isoforms mRNA inhibited cell cleavage in X. laevis embryos, and overexpression of a constitutively active XCaM-KI isoforms resulted abnormal morphology in Hela cells. These results suggest that CaM-KI takes part in cell-structure regulation during early embryonic development. 2. Materials and methods 2.1. cDNA cloning and sequence analysis The X. laevis oocyte cDNA library in lgt10 (Rebagliati et al., 1985) was screened (;3.0=106 clones) using HindIII-SmaI fragment (0.9 kb; amino acid 24–326) of pSG5-hGr (gift from Dr Chatila, Washington University) (Mosialos et al., 1994; Ho et al., 1996) as a probe. Low stringent hybridization was performed for 16 h at 60 8C in a buffer containing 5= SSC, 5= Denhardts’, 0.5% SDS, 100 mgyml salmon sperm DNA, and 50 mM NaPO4 (pH 6.5). Hybridized filters were washed 3 times in a buffer containing 2= SSC and 0.1% SDS at 50 8C. Positive clones were isolated, subcloned into pBluescript II vector (Stratagene), and sequenced by automated procedures. Sequence data were submitted to GenBank娃 (accession number X. laevis CaM-KI LiKb wXCaM-KI LiKbx; AB082999, X. laevis CaMKIawXCaM-KIax; AB083000). 2.2. Construction of plasmids To subclone the full length X. laevis CaM-KI (XCaM-KI) isoform cDNAs into pCS2q or pCSMT (Turner and Weintraub, 1994), the coding regions of XCaM-KI isoforms were amplified by polymerase chain reaction (PCR) using pfu DNA polymerase (Stratagene). The amplified fragments
were digested with EcoRI and XbaI (sites are underlined below), and then ligated into pCS2q or pCSMT. The primers used were as follows: For XCaM-KI LiKb, 59 upstream primer was 59-ggaattccatggcacgggagaacggc-39, the 39 downstream primer was 59-gctctagagctctttacggctggttccatc-39. For XCaM-KIa the 59 upstream primer was 59-ggaattccatgcctctggatgaagatgg-39, and the 39 downstream primer was 59-gctctagagcaggcctcaaactctgttgga-39. Constitutively active mutants of XCaM-KI isoforms were generated by PCR, using a 59 upstream primer similar to that used in amplifying the wild-type isoform, and the 39 downstream primers used were 59-gctctagagcttattgtgcactgacagattcatgg-39 for XCaM-KI LiKb; and 59-gctctagagctagatctgttcgctgactga-39 for XCaMKIa. The resultant constitutively active mutants for XCaM-KI LiKb and XCaM-KIa were named XCaM-KI LiKb (1–297) and XCaM-KIa (1– 295), respectively. Kinase negative mutants were generated using the Quick Change Site-directed mutagenesis kit (Stratagene). The primers used were 59-ccggtaaactttttgctgtggagtgcattccaaagaaagccc-39 to replace lysine 53 with glutamic acid of XCaM-KI LiKb (1–297), and 59-gttggtagccatagagtgtattccaaag-39 to replace lysine 53 with glutamic acid of XCaM-KIa (1–295). The resulting kinase negative mutants were termed XCaM-KI LiKb (1–297, K53E) and XCaM-KIa1-295, K50E), respectively. For in vitro transcription, plasmids were linearized with NotI and transcribed with SP6 RNA polymerase using the mMESSAGE mMACHINE kit (Ambion) according to the manufacturer’s protocols. To generate glutathione-Stransferase (GST)-fusion protein, EcoRI-NotI fragment of XCaM-KI LiKb or XCaM-KIa in the pCS vector were subcloned into pGEX4T-1 (Amersham Pharmacia Biotechnology). 2.3. Antibodies X. laevis CaM-KI antisera were generated by immunization of rabbits with raised synthetic peptide (AISAVGGDRRPRP wamino acids 372–384 of XCaM-KI LiKbx or NSLAYSTHCAQSNRV wamino acids 368–383 of XCaM-KIax) conjugated with keyhole limpet hemocyanin. Antisera were affinity purified on Affigel 10 (Bio-Rad) coupled with immunogen peptides, following the manufacturer’s instruction. The anti-Myc antibody used was 9E10 (Santa Cruz).
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2.4. Western blot analysis Crude lysates of X. laevis embryos were prepared as follows. X. laevis embryos were homogenized in nine volumes of lysis buffer, containing 20 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 10 mM bglycerophosphate, 100 mM Na3VO4, 1 mM NaF, 0.5 mgyml leupeptin, 1 mgyml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 10 000=g for 10 min and supernatants were mixed with 2= SDS-PAGE sample buffer. Samples were boiled for 5 min, subjected to SDS-PAGE, and then transferred to PVDF membrane (Millipore). To detect XCaM-KI proteins, the membrane was probed with antiXCaM-KI LiKb antiserum (1.0 mgyml) or antiXCaM-KIa antiserum (1.0 mgyml), and anti rabbit IgG–HRP (Amersham Pharmacia Biotech) as the secondary antibody (1:2000 dilution) and visualized by an ECL system (Amersham Pharmacia Biotech). 2.5. CaM kinase activity assay Kinase activity was measured at 30 8C for 15 min in 50 ml of 50 mM Tris–Cl (pH 7.5), 0.1 mM Na3VO4, 7 mM NaF, 10 mM MgCl2, 50 mM wg-32Px ATP (1000–2000 cpmypmol), and 40 mM syntide-2 (Bio Mol) containing 1 mM CaCl2, 1 mM calmodulin (CaM) or 1 mM EDTA, 1 mM EGTA. The reaction was terminated by the addition of 20 ml of 4 mM ATP solution. Aliquots (30 ml) were spotted onto phosphocellulose paper (Whatman P-81), followed by washing in 75 mM phosphoric acid. Activities were measured by scintillation counting.
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and incubated for 30 min at room temperature. After washing with the blocking buffer, the membranes were incubated with 1:5000 diluted avidinconjugated horseradish peroxidase (Prozyme) in the blocking buffer. The membranes were washed well with the blocking buffer, and signals were detected with ECL (Amersham Pharmacia Biotech). 2.7. Cell culture and transfection Hela cells (obtained from American Type Culture Collection) were grown in DMEM (Nacalai tesque), supplemented with 10% (vyv) fetal bovine serum, 100 unitsyml penicillin G, 100 mgy ml streptomycin, and 2 mM L-glutamine. Cells were grown in a 5% CO2, 95% air humidified atmosphere. Hela cells were transiently transfected using Trans IT LT1 reagent (Mirus) according to the manufacturer’s instructions. 2.8. Immunofluorescence staining Transfected Hela cells were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. Myc-tagged XCaM-KI mutants were stained with anti-Myc antibody, 9E10 (0.25 mgyml, Santa Cruz). The second antibody was Alexa488-labeled anti mouse IgG (1:500 dilution, Molecular Probes). 2.9. Others X. laevis embryo manipulations were performed as described (Saneyoshi et al., 2002). Protein concentration was determined by Bradford dye assay (Bio-Rad) with bovine serum albumin as protein standard.
2.6. Biotinylated-CaM overlay assay 3. Results The biotinylated-CaM overlay assay was as described previously (Saneyoshi et al., 2000). GST-fusion recombinant XCaM-KI mutants were resolved on 10% SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Millipore). The membrane was blocked with the blocking buffer (0.1 M NaCl, 50 mM Tris–Cl (pH 7.5), 0.1% bovine serum albumin, 0.05% Tween 20) in the presence of 1 mM CaCl2 or 2 mM EGTA for 30 min at room temperature. Biotinylated-CaM (Calbiochem) was added at a final concentration of 0.5 mgyml in the blocking buffer
3.1. Isolation and characterization of cDNA clones for the XCaM-KI isoforms In order to obtain X. laevis Ca2q ycalmodulindependent protein kinase (CaM-K) cDNA, we screened a X. laevis oocyte cDNA library (Rebagliati et al., 1985), using a catalytic domain of human CaM kinase IV (amino acid 24–326) (Ho et al., 1996) as a probe. The obtained clones exhibited high homology with CaM-KI. The clone XGr17 was 1419 bp, containing a 383 amino acid
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Fig. 1. Alignment of X. laevis and human CaM-KI isoforms. Amino acid sequence comparison of X. laevis CaM-KIs with human CaMKIs. (accession number; human CaM-KIa, L41816; human CaM-KI like kinase, AF286366). The respective amino acid numbers are shown at the left. Amino acid residues identical to those of the X. laevis homolog are boxed. The catalytic domain is indicated by a solid line box. The phosphorylation site Thr181 in XCaM-KI LiKb or Thr178 in XCaM-KIa responsible for activation by CaM-KK, equivalent to Thr177 in human CaM-KIa (Haribabu et al., 1995) or Thr180 in human CaM-KI LiK (Verploegen et al., 2000) are indicated by an asterisk. The regulatory region containing the AID and CBD is indicated by a solid line and a broken line, respectively. The nucleotide sequences of XCaM-KI LiKb and XCaMKIa have been deposited in the DDBJyEMBLyGenBank娃 DNA database under the accession number AB082999 and AB083000, respectively.
open reading frame that was 79.4% identical at the amino acid level to human CaM-KIa (Haribabu et al., 1995); it was named X. laevis CaMKI like kinase XCaM-KIa. The other clones, XGr3 and 6, were 2107 bp containing a 396 amino acid open reading frame, and they were identical independent clones. XGr3y6 showed the strongest overall sequence similarity to human CaM-KI like kinase (CaM-KI LiK) (Verploegen et al., 2000), but with a distinct C-terminal sequence. Therefore, XGr3y6 was a new isoform of CaM-KI, and it was named X. laevis homologue of b-isoform CaM-KI LiK (XCaM-KI LiKb). CaM-KI LiKb was also found in humans (accession number,
AX167585), suggesting that CaM-KI LiKb is a well-conserved molecule among vertebrates. The deduced amino acid sequences of human and X. laevis CaM-KI are shown in Fig. 1. The predicted molecular mass of the full-length peptide of XCaM-KI LiKb was 43.8 kDa, while XCaM-KIa was 43.0 kDa, in reasonable agreement with the value of ;45 kDa estimated by western blot analysis, as shown in Fig. 3. 3.2. XCaM-KI isoforms are expressed at early stages of embryonic development To determine the temporal expression of XCaMKI LiKb and XCaM-KIa protein during X. laevis
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embryos and Hela cells (data not shown). XCaMKIa was constantly expressed throughout the early embryonic stages at substantial levels. In addition, whereas the expression level of XCaM-KI LiKb was initially low during the cleavage stages, it increased gradually after the neurula stage and was maintained at a substantial level thereafter (Fig. 2a). We next analyzed the distribution of both XCaM-KI isoforms in adult tissues (Fig. 2b). XCaM-KI LiKb protein was ubiquitously expressed in adult tissues, and was particularly abundant in the brain and testis. XCaM-KIa protein was expressed mainly in the brain, heart, skeletal muscle, and oocytes (Fig. 2b). The band observed in the brain in both XCaM-KI isoforms appeared in doublet, which is likely due to phosphorylation. These results suggest that the two XCaM-KI isoforms have different roles in early development and distinct functions in different tissues. 3.3. Both isoforms of XCaM-KI are regulated by Ca2q yCaM
Fig. 2. Expression profile of XCaM-KI proteins. (a) Temporal expression of XCaM-KI LiKb and XCaM-KIa proteins in early embryonic development. Fifty micrograms of crude lysates from indicated stages of embryos were loaded onto each lane. The blotted filters were probed with an anti-XCaM-KI LiKb antibody (top) or an anti-XCaM-KIa antibody (bottom). UFE, unfertilized egg. Staging was according to Nieuwkoop and Faber (1967) (Nieuwkoop and Faber, 1967); (b) Tissue distributions of XCaM-KI LiKb and XCaM-KIa protein. One hundred micrograms of crude lysates were loaded onto each lane. The blotted filters were probed with an anti-XCaM-KI LiKb antibody (top) or an anti-XCaM-KIa antibody (bottom). (a) and (b), Molecular mass markers are indicated on the left (kDa).
development, Western blot analysis was performed (Fig. 2a). We generated specific antibodies against XCaM-KI LiKb and XCaM-KIa using C-terminal synthetic peptides in each isoform as immunogens. The specificities of these antibodies were confirmed by their reactivities to the transient expression of both of XCaM-KI isoforms in X. laevis
The primary structure of XCaM-KI indicates that both isoforms consist of a putative CaM binding domain (CBD) and an auto-inhibitory domain (AID) (Fig. 1) (Soderling and Stull, 2001). To test whether the XCaM-KI isoforms were regulated by Ca2q yCaM, we performed a CaM-overlay assay and kinase assay. We constructed GST-fusion proteins of both wild-types and the deletion mutants that lacked both the CBD and the AID. The resulting construct for XCaM-KI LiKb consisted of amino acid 1–297 and the construct for XCaM-KIa consisted of amino acid 1–295. Both wild-type XCaM-KI isoforms were capable of binding to CaM, whereas the deletion mutants were incapable of CaM binding in the presence of calcium (Fig. 3a). Next, with an in vitro phosphorylation assay using a known peptide substrate, we determined whether XCaM-KI LiKb and XCaM-KIa had Ca2q yCaM-dependent kinase activities. Fig. 3b shows that both isoforms of XCaM-KI were able to phosphorylate syntide-2 peptide in a Ca2q y CaM-dependent manner. Although wild-type CaMKI was inactive in the absence of Ca2q yCaM, the deletion mutants—XCaM-KI LiKb (1–297) and XCaM-KIa (1–295), which lacked both CBD and AID—were active regardless of Ca2q yCaM. These results are consistent with the previous reports in
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these deletion mutants act as constitutively active kinases (Fig. 3b). CaM-KI is known to be activated by upstream Ca2q yCaM-dependent kinase (CaM-KK) (Tokumitsu et al., 1994; Soderling and Stull, 2001). The mechanism of this activation by CaM-KK is due to phosphorylation of a Thr residue within the Tloop in CaM-KI (Goldberg et al., 1996). The corresponding Thr residues Thr181 and Thr178 are conserved in XCaM-KI LiKb and XCaM-KIa, respectively (Fig. 1). To test whether XCaM-KI regulates CaM-KK on Thr181 or Thr178, we constructed mutants with a Thr-to-Asp substitution at the putative CaM-KK phosphorylation site. The mutants were named XCaM-KI LiKb T181D or XCaM-KIa T178D. The substitution at the phosphorylation site was predicted to mimic phosphorylation, and indeed, XCaM-KI LiKb T181D and XCaM-KIa T178D both showed increased kinase activities compared with that of the wild-type, which is consistent with previously reported findings in mammalian CaM-KI and CaM-KIV (Tokumitsu et al., 1994; Haribabu et al., 1995). These results suggest that XCaM-KI LiKb and XCaMKIa were regulated by an upstream kinase, probably X. laevis CaM-KK (Wayman et al., 2000). 3.4. Constitutively active XCaM-KI inhibits cell cleavage
Fig. 3. CaM binding activities and kinase activities of recombinant XCaM-KIs. (a) Purified GST-fusion XCaM-KIs were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R250, or blotted onto a PVDF membrane (Ca2q or EDTA). The blots were reacted with a biotinylatedCaM in the presence of either 1 mM CaCl2 (Ca2q) or 1 mM EDTA (EDTA). GST-XCaM-KI LiKß wild-type and GSTXCaM-KI LiKb (1–297) were indicate as WT and 1–297, respectively. GST-XCaM-KIa wild-type and GST-XCaM-KIa (1–295) were indicate as WT and 1–295, respectively. Molecular mass markers are indicated on the left (kDa). Both wildtype XCaM-KI isoforms were capable of binding to CaM, whereas deletion mutants were incapable of CaM binding in the presence of calcium; (b) Kinase activities of both GSTfusion XCaM-KI isoforms were measured at 30 8C for 15 min. Values are mean"S.E.M. of triplicate experiments, each done in duplicate.
human CaM-KI (Haribabu et al., 1995; Verploegen et al., 2000), confirming that these two XCaM-KI isoforms are regulated by Ca2q yCaM and that
In order to examine the role of CaM-KI in X. laevis early development, we injected various mutants of XCaM-KI mRNA into X. laevis embryos. Both constitutively active mutants of XCaMKI, XCaM-KI LiKb (1–297) and XCaM-KIa (1–295), inhibited cell cleavage (Fig. 4a, constitutively active mutant). As expected from the homology with human CaM-KI (Haribabu et al., 1995), replacement of the essential lysine at the ATP-binding site to a glutamic acid (Lysine 53 for XCaM-KI LiKb, or Lysine 50 for XCaM-KIa) resulted in inactive enzymes in vitro (data not shown). Injection of this kinase negative mutant (Fig. 4a, kinase negative mutant: XCaM-KI LiKb w1–297, K53Ex or XCaM-KIa w1–295, K50Ex) did not show cell cleavage arrest, thereby confirming that cell cleavage arrest was caused specifically by the activated kinase activities of XCaM-KI. (The cleavage arrest observed here is not likely through transcription, since major zygotic transcription starts after mid-blastula transition at approximately stage 9.)
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that both CaM-KI isoforms are cytosolic proteins (Soderling and Stull, 2001). Both constitutively active mutants of XCaM-KI (XCaM-KI LiKb w1– 297x or XCaM-KIa w1–295x) caused abnormal morphology when transfected into Hela cells (Fig. 4b, middle panel, constitutively active mutant). As kinase negative mutants (XCaM-KI LiKb w1–297, K53Ex or XCaM-KIa w1–295, K50Ex) gave normal cell morphology in transfected cells (Fig. 4b, bottom panel, kinase negative mutant), this indicates that the cell abnormality observed was caused specifically by the constitutive kinase activity of XCaM-KI. 4. Discussion
Fig. 4. The effects of XCaM-KI in X. laevis embryos and cultured cells. (a) Overexpression of constitutively active CaMKI inhibited cell cleavage in X. laevis embryos. RNA encoding XCaM-KI LiKb (left, 125 pg) or XCaM-KIa (right, 62.5 pg) was injected into a single blastomere at 2-cell stage embryos. Injected embryos were cultured until stage 8. Constitutively active mutant, top panels; kinase negative mutant, bottom panels; (b) Subcellular localization and effects of XCaM-KI isoforms in Hela cells. Hela cells were transiently transfected in 6-well plates with 1 mg of pCS2q plasmids expressing Myctagged wild-types or mutants XCaM-KI isoforms. Wild-type XCaM-KI LiKb or XCaM-KIa (top, wild-type); XCaM-KI LiKb (1–297) or XCaM-KIa (1–295) (middle, constitutively active mutant); XCaM-KI LiKb (1–297, K53E) or XCaMKIa (1–295, K50E) (bottom, kinase negative mutant). After 24 h, the transfected cells were fixed, and immunofluorostained with an anti-Myc monoclonal antibody (9E10). Constitutively active mutants of both XCaM-KI isoforms caused abnormal morphology in Hela cells.
We next examined the subcellular localization of XCaM-KI isoforms and their functions in cultured cells. Both wild-type XCaM-KI LiKb and wild-type XCaM-KIa localized mainly in the cytoplasm (Fig. 4b, top panel, wild-type), confirming
We have shown that at least two different isoforms of CaM-KI are expressed in X. laevis early embryogenesis. These have been designated XCaM-KI LiKb and XCaM-KIa. When the cell cleavage progresses in X. laevis embryos, a transient rise of wCa2qxi is observed along the furrow (Muto et al., 1996). While it is known that cleavage furrow formation requires IP3 receptormediated Ca2q signaling (Mitsuyama et al., 1999), the role of Ca2q and its downstream targets are unclear. But we found that XCaM-KIa protein expressed throughout early cleavage stages (Fig. 2a), and it was activated by Ca2q yCaM (Fig. 3b). Moreover, overexpression of a constitutively active mutant of CaM-KI isoforms caused cell cleavage arrest in X. laevis embryos (Fig. 4a), while overexpression of either constitutively active CaM-KII or CaM-KIV, which are other multi-functional CaM-K, does not inhibit cell cleavage in X. laevis ¨ et al., 2000; Wayman et al., 2000). embryos (Kuhl Therefore, XCaM-KIa may have a role in cell cleavage progression. The cleavage arrest phenotype is also observed in the activation of the MAPK signaling pathway in X. laevis embryos (Haccard et al., 1993; Kosako et al., 1994; Bhatt and Ferrell, 1999; Gross et al., 1999). Moreover, the MAPK pathway is activated not only by the calcium ionophore (Franklin et al., 2000) but also by CaM-K (Enslen et al., 1996; Verploegen et al., 2000). We therefore tested the possibility of the activation of the MAPK pathway by XCaM-KI. However, neither XCaM-KI LiKb (1–297) nor XCaM-KIa (1–295) could activate MAPK in embryos, nor did they cause germinal vesicle break down in the X. laevis oocytes (T.S.
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unpublished results). Therefore, XCaM-KI is unlikely to activate the MAPK signaling pathway. We also studied whether CaM-KI had a role in microtubule dynamics, as has been shown for CaM-KIV. CaM-KIV regulates microtubule dynamics through phosphorylation oncoprotein 18 on Ser 16 (Marklund et al., 1994; Melander Gradin et al., 1997). Considering the substrate similarities between CaM-KI and -KIV, it is possible that XCaM-KI also regulates microtubule dynamics. Overexpression of constitutively active XCaM-KI mutants, however, had no effect on gross cellular distribution of tubulin protein in Hela cells (T.S. unpublished result), thereby showing that tubulin might not be the downstream substrate for XCaMKI. Muto and Mikoshiba have reported that a transient increase of Ca2q induces cell shape changes in fertilized eggs and that the contracted region is strongly stained with rhodamine-phalloidin (Muto and Mikoshiba, 1998). Taken together, our results suggest that cytoskeletal proteins, e.g. F-actin, may be candidate targets regulated by Ca2q through the action of CaM-KI. Of additional interest is that the CaM-KI LiK amino acid in humans and the XCaM-KI LiKb amino acid in X. laevis is almost the same, except for the C-terminal portion. The human homologue of CaM-KI LiKb (GenBank accession number AX167585) shows very high similarities with human CaM-KI LiK, which is 97.5% identical at the amino acid level. It is possible these two isoforms are alternatively spliced variants, as are CaM-KI b1 and b2 (Naito et al., 1997). At present, X. laevis CaM-KI LiK and human CaMKI LiKb have been not reported; however, human CaM-KI LiK highly expresses in polymorphonuclear leukocytes (Verploegen et al., 2000), while XCaM-KI LiKb highly expressed in brain and testis (Fig. 2b). These differences suggest that the C-terminal sequence of CaM-KI LiK may have an important role for isoform-specific characteristics or tissue distribution. Future studies will examine the endogenous substrate of XCaM-KI LiKb, and its functional consequences. Acknowledgments We thank Drs T. Chatila, D. Melton and D. Turner, for the reagents. We thank Drs A. Mizutani and H. Tokumitsu for their critical discussion of the manuscript, and Y. Takeyama for technical
assistance. T. S. was supported by the JSPS Research Fellowships for Young Scientists. References Bhatt, R.R., Ferrell Jr., J.E., 1999. The protein kinase p90 rsk as an essential mediator of cytostatic factor activity. Science 286, 1362–1365. Enslen, H., Tokumitsu, H., Stork, P.J., Davis, R.J., Soderling, T.R., 1996. Regulation of mitogen-activated protein kinases by a calciumycalmodulin-dependent protein kinase cascade. Proc. Natl. Acad. Sci. USA 93, 10803–10808. Franklin, R.A., Atherfold, P.A., McCubrey, J.A., 2000. Calcium-induced ERK activation in human T lymphocytes occurs via p56 (Lck) and CaM-kinase. Mol. Immunol. 37, 675–683. Goldberg, J., Nairn, A.C., Kuriyan, J., 1996. Structural basis for the autoinhibition of calciumycalmodulin-dependent protein kinase I. Cell 84, 875–887. Gross, S.D., Schwab, M.S., Lewellyn, A.L., Maller, J.L., 1999. Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Science 286, 1365–1367. Haccard, O., Sarcevic, B., Lewellyn, A., et al., 1993. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science 262, 1262–1265. Haribabu, B., Hook, S.S., Selbert, M.A., et al., 1995. Human calcium–calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium–calmodulin dependent protein kinase I kinase. EMBO J. 14, 3679–3686. Ho, N., Gullberg, M., Chatila, T., 1996. Activation protein 1dependent transcriptional activation of interleukin 2 gene by Ca2qycalmodulin kinase type IVyGr. J. Exp. Med. 184, 101–112. Kosako, H., Gotoh, Y., Nishida, E., 1994. Mitogen-activated protein kinase is required for the mos-induced metaphase arrest. J. Biol. Chem. 269, 28354–28358. ¨ Kuhl, M., Sheldahl, L.C., Malbon, C.C., Moon, R.T., 2000. Ca2qycalmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J. Biol. Chem. 275, 12701–12711. Kume, S., 1999. Role of the inositol 1,4,5-trisphosphate receptor in early embryonic development. Cell Mol. Life Sci. 56, 296–304. Kume, S., Muto, A., Inoue, T., Suga, K., Okano, H., Mikoshiba, K., 1997. Role of inositol 1,4,5-trisphosphate receptor in ventral signaling in Xenopus embryos. Science 278, 1940–1943. Lorca, T., Cruzalegui, F.H., Fesquet, D., et al., 1993. Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature 366, 270–273. Marklund, U., Larsson, N., Brattsand, G., Osterman, O., Chatila, T.A., Gullberg, M., 1994. Serine 16 of oncoprotein 18 is a major cytosolic target for the Ca2q ycalmodulindependent kinase-Gr. Eur. J. Biochem. 225, 53–60. Means, A.R., 2000. Regulatory cascades involving calmodulindependent protein kinases. Mol. Endocrinol. 14, 4–13. Melander Gradin, H., Marklund, U., Larsson, N., Chatila, T.A., Gullberg, M., 1997. Regulation of microtubule dynamics by
T. Saneyoshi et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 499–507 Ca2qycalmodulin-dependent kinase IVyGr-dependent phosphorylation of oncoprotein 18. Mol. Cell. Biol. 17, 3459–3467. Mitsuyama, F., Sawai, T., Carafoli, E., Furuichi, T., Mikoshiba, K., 1999. Microinjection of Ca2q store-enriched microsome fractions to dividing newt eggs induces extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2q release. Dev. Biol. 214, 160–167. Morin, N., Abrieu, A., Lorca, T., Martin, F., Doree, M., 1994. The proteolysis-dependent metaphase to anaphase transition: calciumycalmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs. EMBO J. 13, 4343–4352. Mosialos, G., Hanissian, S.H., Jawahar, S., Vara, L., Kieff, E., Chatila, T.A., 1994. A Ca2q ycalmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein–Barr virus (EBV) is induced by the EBV oncogene LMP1. J. Virol. 68, 1697–1705. Muto, A., Kume, S., Inoue, T., Okano, H., Mikoshiba, K., 1996. Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin. J. Cell Biol. 135, 181–190. Muto, A., Mikoshiba, K., 1998. Activation of inositol 1,4,5trisphosphate receptors induces transient changes in cell shape of fertilized Xenopus eggs. Cell Motil. Cytoskel. 39, 201–208. Naito, Y., Watanabe, Y., Yokokura, H., Sugita, R., Nishio, M., Hidaka, H., 1997. Isoform-specific activation and structural diversity of calmodulin kinase I. J. Biol. Chem. 272, 32704–32708.
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Calciumycalmodulin-dependent protein kinase I in Xenopus laevis夞 Takeo Saneyoshia,*, Shoen Kumeb, Katsuhiko Mikoshibaa,c,d a Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama, Wako-shi 351-0198, Japan Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan c Department of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan d Calcium Oscillation Project, ICORP, Japan Science and Technology Corporation (JST), 3-14-4 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
b
Received 29 July 2002; received in revised form 5 November 2002; accepted 11 December 2002
Abstract Calciumycalmodulin (CaM) dependent protein kinase I (CaM-KI) is a member of a well-defined multi-functional CaM-K family, but its physiological and developmental functions have yet to be determined. Here, we have cloned two cDNAs encoding CaM-KI from a Xenopus laevis (X. laevis) oocyte cDNA library. One is a novel isoform of CaM-KI, named CaM-KI LiKb (XCaM-KI LiKb). The other is an a isoform of CaM-KI (XCaM-KIa), which is a highly related to previously cloned mammalian isoform. XCaM-KIa was constantly expressed through embryogenesis, whereas XCaMKI LiKb expression dramatically increased in the neurula stage. Both XCaM-KI isoforms exhibited kinase activity in a Ca2qyCaM-dependent manner. Overexpression of a constitutively active mutant of CaM-KI isoforms inhibited cell cleavage in X. laevis embryos and caused a marked change of cell morphology in Hela cells. Taken together, these results suggest that CaM-KI plays a role in cell-structure regulation during early embryonic development. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Calcium; Calmodulin; Protein kinase; Xenopus laevis; cDNA cloning; Novel isoform; Cell cleavage arrest
1. Introduction In early embryogenesis, the inositol 1,4,5-trisphosphate (IP3)–Ca2q signal transduction pathway is involved in many biological processes, including fertilization, cell cleavage, and dorsoventral axis formation (Kume et al., 1997; Kume, 1999; Saneyoshi et al., 2002). The participation of 夞 The nucleotide sequences of XCaM-KI LiKb and XCaMKIa have been deposited in DDBJyEMBLyGenBank娃 DNA database under the accession numbers AB082999 and AB083000, respectively. *Corresponding author. Tel.: q81-484679745; fax: q81484679744. E-mail address: [email protected] (T. Saneyoshi).
Ca2q in cell division has been proposed, but its molecular mechanism is not well understood (Muto et al., 1996; Muto and Mikoshiba, 1998; Mitsuyama et al., 1999). The Ca2q ycalmodulin-dependent protein kinase (CaM-K) family has three known related members: CaM-KI, -KII and -KIV (Soderling and Stull, 2001). CaM-KI and IV are closely related protein kinases with many biochemical similarities, and they are regulated by CaM-K kinase (CaM-KK), which is further upstream, through phosphorylation of the Thr residue in the activation loop in vitro, —also a Ca2q ycalmodulin-dependent event. Though CaM-KII and CaM-KIV have been associated with many aspects of cellular functions, the
1096-4959/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959(02)00292-0
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physiological and developmental role of CaM-KI is still unclear (Lorca et al., 1993; Morin et al., ¨ et al., 2000; Means, 2000; Wayman et 1994; Kuhl al., 2000; Soderling et al., 2001). In this paper, we report molecular cloning of two isoforms of Xenopus laevis (X. laevis) CaMKI (XCaM-KI), one is an a isoform of CaM-KI (XCaM-KIa), and the other is a novel isoform of CaM-KI, which named XCaM-KI LiKb. Our data demonstrate that both XCaM-KI isoforms conformed to the properties of the mammalian CaMKI, and the expression of the two XCaM-KI isoform was differentially regulated. Moreover, we show that injection of a constitutively active XCaM-KI isoforms mRNA inhibited cell cleavage in X. laevis embryos, and overexpression of a constitutively active XCaM-KI isoforms resulted abnormal morphology in Hela cells. These results suggest that CaM-KI takes part in cell-structure regulation during early embryonic development. 2. Materials and methods 2.1. cDNA cloning and sequence analysis The X. laevis oocyte cDNA library in lgt10 (Rebagliati et al., 1985) was screened (;3.0=106 clones) using HindIII-SmaI fragment (0.9 kb; amino acid 24–326) of pSG5-hGr (gift from Dr Chatila, Washington University) (Mosialos et al., 1994; Ho et al., 1996) as a probe. Low stringent hybridization was performed for 16 h at 60 8C in a buffer containing 5= SSC, 5= Denhardts’, 0.5% SDS, 100 mgyml salmon sperm DNA, and 50 mM NaPO4 (pH 6.5). Hybridized filters were washed 3 times in a buffer containing 2= SSC and 0.1% SDS at 50 8C. Positive clones were isolated, subcloned into pBluescript II vector (Stratagene), and sequenced by automated procedures. Sequence data were submitted to GenBank娃 (accession number X. laevis CaM-KI LiKb wXCaM-KI LiKbx; AB082999, X. laevis CaMKIawXCaM-KIax; AB083000). 2.2. Construction of plasmids To subclone the full length X. laevis CaM-KI (XCaM-KI) isoform cDNAs into pCS2q or pCSMT (Turner and Weintraub, 1994), the coding regions of XCaM-KI isoforms were amplified by polymerase chain reaction (PCR) using pfu DNA polymerase (Stratagene). The amplified fragments
were digested with EcoRI and XbaI (sites are underlined below), and then ligated into pCS2q or pCSMT. The primers used were as follows: For XCaM-KI LiKb, 59 upstream primer was 59-ggaattccatggcacgggagaacggc-39, the 39 downstream primer was 59-gctctagagctctttacggctggttccatc-39. For XCaM-KIa the 59 upstream primer was 59-ggaattccatgcctctggatgaagatgg-39, and the 39 downstream primer was 59-gctctagagcaggcctcaaactctgttgga-39. Constitutively active mutants of XCaM-KI isoforms were generated by PCR, using a 59 upstream primer similar to that used in amplifying the wild-type isoform, and the 39 downstream primers used were 59-gctctagagcttattgtgcactgacagattcatgg-39 for XCaM-KI LiKb; and 59-gctctagagctagatctgttcgctgactga-39 for XCaMKIa. The resultant constitutively active mutants for XCaM-KI LiKb and XCaM-KIa were named XCaM-KI LiKb (1–297) and XCaM-KIa (1– 295), respectively. Kinase negative mutants were generated using the Quick Change Site-directed mutagenesis kit (Stratagene). The primers used were 59-ccggtaaactttttgctgtggagtgcattccaaagaaagccc-39 to replace lysine 53 with glutamic acid of XCaM-KI LiKb (1–297), and 59-gttggtagccatagagtgtattccaaag-39 to replace lysine 53 with glutamic acid of XCaM-KIa (1–295). The resulting kinase negative mutants were termed XCaM-KI LiKb (1–297, K53E) and XCaM-KIa1-295, K50E), respectively. For in vitro transcription, plasmids were linearized with NotI and transcribed with SP6 RNA polymerase using the mMESSAGE mMACHINE kit (Ambion) according to the manufacturer’s protocols. To generate glutathione-Stransferase (GST)-fusion protein, EcoRI-NotI fragment of XCaM-KI LiKb or XCaM-KIa in the pCS vector were subcloned into pGEX4T-1 (Amersham Pharmacia Biotechnology). 2.3. Antibodies X. laevis CaM-KI antisera were generated by immunization of rabbits with raised synthetic peptide (AISAVGGDRRPRP wamino acids 372–384 of XCaM-KI LiKbx or NSLAYSTHCAQSNRV wamino acids 368–383 of XCaM-KIax) conjugated with keyhole limpet hemocyanin. Antisera were affinity purified on Affigel 10 (Bio-Rad) coupled with immunogen peptides, following the manufacturer’s instruction. The anti-Myc antibody used was 9E10 (Santa Cruz).
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2.4. Western blot analysis Crude lysates of X. laevis embryos were prepared as follows. X. laevis embryos were homogenized in nine volumes of lysis buffer, containing 20 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 10 mM bglycerophosphate, 100 mM Na3VO4, 1 mM NaF, 0.5 mgyml leupeptin, 1 mgyml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 10 000=g for 10 min and supernatants were mixed with 2= SDS-PAGE sample buffer. Samples were boiled for 5 min, subjected to SDS-PAGE, and then transferred to PVDF membrane (Millipore). To detect XCaM-KI proteins, the membrane was probed with antiXCaM-KI LiKb antiserum (1.0 mgyml) or antiXCaM-KIa antiserum (1.0 mgyml), and anti rabbit IgG–HRP (Amersham Pharmacia Biotech) as the secondary antibody (1:2000 dilution) and visualized by an ECL system (Amersham Pharmacia Biotech). 2.5. CaM kinase activity assay Kinase activity was measured at 30 8C for 15 min in 50 ml of 50 mM Tris–Cl (pH 7.5), 0.1 mM Na3VO4, 7 mM NaF, 10 mM MgCl2, 50 mM wg-32Px ATP (1000–2000 cpmypmol), and 40 mM syntide-2 (Bio Mol) containing 1 mM CaCl2, 1 mM calmodulin (CaM) or 1 mM EDTA, 1 mM EGTA. The reaction was terminated by the addition of 20 ml of 4 mM ATP solution. Aliquots (30 ml) were spotted onto phosphocellulose paper (Whatman P-81), followed by washing in 75 mM phosphoric acid. Activities were measured by scintillation counting.
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and incubated for 30 min at room temperature. After washing with the blocking buffer, the membranes were incubated with 1:5000 diluted avidinconjugated horseradish peroxidase (Prozyme) in the blocking buffer. The membranes were washed well with the blocking buffer, and signals were detected with ECL (Amersham Pharmacia Biotech). 2.7. Cell culture and transfection Hela cells (obtained from American Type Culture Collection) were grown in DMEM (Nacalai tesque), supplemented with 10% (vyv) fetal bovine serum, 100 unitsyml penicillin G, 100 mgy ml streptomycin, and 2 mM L-glutamine. Cells were grown in a 5% CO2, 95% air humidified atmosphere. Hela cells were transiently transfected using Trans IT LT1 reagent (Mirus) according to the manufacturer’s instructions. 2.8. Immunofluorescence staining Transfected Hela cells were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. Myc-tagged XCaM-KI mutants were stained with anti-Myc antibody, 9E10 (0.25 mgyml, Santa Cruz). The second antibody was Alexa488-labeled anti mouse IgG (1:500 dilution, Molecular Probes). 2.9. Others X. laevis embryo manipulations were performed as described (Saneyoshi et al., 2002). Protein concentration was determined by Bradford dye assay (Bio-Rad) with bovine serum albumin as protein standard.
2.6. Biotinylated-CaM overlay assay 3. Results The biotinylated-CaM overlay assay was as described previously (Saneyoshi et al., 2000). GST-fusion recombinant XCaM-KI mutants were resolved on 10% SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Millipore). The membrane was blocked with the blocking buffer (0.1 M NaCl, 50 mM Tris–Cl (pH 7.5), 0.1% bovine serum albumin, 0.05% Tween 20) in the presence of 1 mM CaCl2 or 2 mM EGTA for 30 min at room temperature. Biotinylated-CaM (Calbiochem) was added at a final concentration of 0.5 mgyml in the blocking buffer
3.1. Isolation and characterization of cDNA clones for the XCaM-KI isoforms In order to obtain X. laevis Ca2q ycalmodulindependent protein kinase (CaM-K) cDNA, we screened a X. laevis oocyte cDNA library (Rebagliati et al., 1985), using a catalytic domain of human CaM kinase IV (amino acid 24–326) (Ho et al., 1996) as a probe. The obtained clones exhibited high homology with CaM-KI. The clone XGr17 was 1419 bp, containing a 383 amino acid
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Fig. 1. Alignment of X. laevis and human CaM-KI isoforms. Amino acid sequence comparison of X. laevis CaM-KIs with human CaMKIs. (accession number; human CaM-KIa, L41816; human CaM-KI like kinase, AF286366). The respective amino acid numbers are shown at the left. Amino acid residues identical to those of the X. laevis homolog are boxed. The catalytic domain is indicated by a solid line box. The phosphorylation site Thr181 in XCaM-KI LiKb or Thr178 in XCaM-KIa responsible for activation by CaM-KK, equivalent to Thr177 in human CaM-KIa (Haribabu et al., 1995) or Thr180 in human CaM-KI LiK (Verploegen et al., 2000) are indicated by an asterisk. The regulatory region containing the AID and CBD is indicated by a solid line and a broken line, respectively. The nucleotide sequences of XCaM-KI LiKb and XCaMKIa have been deposited in the DDBJyEMBLyGenBank娃 DNA database under the accession number AB082999 and AB083000, respectively.
open reading frame that was 79.4% identical at the amino acid level to human CaM-KIa (Haribabu et al., 1995); it was named X. laevis CaMKI like kinase XCaM-KIa. The other clones, XGr3 and 6, were 2107 bp containing a 396 amino acid open reading frame, and they were identical independent clones. XGr3y6 showed the strongest overall sequence similarity to human CaM-KI like kinase (CaM-KI LiK) (Verploegen et al., 2000), but with a distinct C-terminal sequence. Therefore, XGr3y6 was a new isoform of CaM-KI, and it was named X. laevis homologue of b-isoform CaM-KI LiK (XCaM-KI LiKb). CaM-KI LiKb was also found in humans (accession number,
AX167585), suggesting that CaM-KI LiKb is a well-conserved molecule among vertebrates. The deduced amino acid sequences of human and X. laevis CaM-KI are shown in Fig. 1. The predicted molecular mass of the full-length peptide of XCaM-KI LiKb was 43.8 kDa, while XCaM-KIa was 43.0 kDa, in reasonable agreement with the value of ;45 kDa estimated by western blot analysis, as shown in Fig. 3. 3.2. XCaM-KI isoforms are expressed at early stages of embryonic development To determine the temporal expression of XCaMKI LiKb and XCaM-KIa protein during X. laevis
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embryos and Hela cells (data not shown). XCaMKIa was constantly expressed throughout the early embryonic stages at substantial levels. In addition, whereas the expression level of XCaM-KI LiKb was initially low during the cleavage stages, it increased gradually after the neurula stage and was maintained at a substantial level thereafter (Fig. 2a). We next analyzed the distribution of both XCaM-KI isoforms in adult tissues (Fig. 2b). XCaM-KI LiKb protein was ubiquitously expressed in adult tissues, and was particularly abundant in the brain and testis. XCaM-KIa protein was expressed mainly in the brain, heart, skeletal muscle, and oocytes (Fig. 2b). The band observed in the brain in both XCaM-KI isoforms appeared in doublet, which is likely due to phosphorylation. These results suggest that the two XCaM-KI isoforms have different roles in early development and distinct functions in different tissues. 3.3. Both isoforms of XCaM-KI are regulated by Ca2q yCaM
Fig. 2. Expression profile of XCaM-KI proteins. (a) Temporal expression of XCaM-KI LiKb and XCaM-KIa proteins in early embryonic development. Fifty micrograms of crude lysates from indicated stages of embryos were loaded onto each lane. The blotted filters were probed with an anti-XCaM-KI LiKb antibody (top) or an anti-XCaM-KIa antibody (bottom). UFE, unfertilized egg. Staging was according to Nieuwkoop and Faber (1967) (Nieuwkoop and Faber, 1967); (b) Tissue distributions of XCaM-KI LiKb and XCaM-KIa protein. One hundred micrograms of crude lysates were loaded onto each lane. The blotted filters were probed with an anti-XCaM-KI LiKb antibody (top) or an anti-XCaM-KIa antibody (bottom). (a) and (b), Molecular mass markers are indicated on the left (kDa).
development, Western blot analysis was performed (Fig. 2a). We generated specific antibodies against XCaM-KI LiKb and XCaM-KIa using C-terminal synthetic peptides in each isoform as immunogens. The specificities of these antibodies were confirmed by their reactivities to the transient expression of both of XCaM-KI isoforms in X. laevis
The primary structure of XCaM-KI indicates that both isoforms consist of a putative CaM binding domain (CBD) and an auto-inhibitory domain (AID) (Fig. 1) (Soderling and Stull, 2001). To test whether the XCaM-KI isoforms were regulated by Ca2q yCaM, we performed a CaM-overlay assay and kinase assay. We constructed GST-fusion proteins of both wild-types and the deletion mutants that lacked both the CBD and the AID. The resulting construct for XCaM-KI LiKb consisted of amino acid 1–297 and the construct for XCaM-KIa consisted of amino acid 1–295. Both wild-type XCaM-KI isoforms were capable of binding to CaM, whereas the deletion mutants were incapable of CaM binding in the presence of calcium (Fig. 3a). Next, with an in vitro phosphorylation assay using a known peptide substrate, we determined whether XCaM-KI LiKb and XCaM-KIa had Ca2q yCaM-dependent kinase activities. Fig. 3b shows that both isoforms of XCaM-KI were able to phosphorylate syntide-2 peptide in a Ca2q y CaM-dependent manner. Although wild-type CaMKI was inactive in the absence of Ca2q yCaM, the deletion mutants—XCaM-KI LiKb (1–297) and XCaM-KIa (1–295), which lacked both CBD and AID—were active regardless of Ca2q yCaM. These results are consistent with the previous reports in
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these deletion mutants act as constitutively active kinases (Fig. 3b). CaM-KI is known to be activated by upstream Ca2q yCaM-dependent kinase (CaM-KK) (Tokumitsu et al., 1994; Soderling and Stull, 2001). The mechanism of this activation by CaM-KK is due to phosphorylation of a Thr residue within the Tloop in CaM-KI (Goldberg et al., 1996). The corresponding Thr residues Thr181 and Thr178 are conserved in XCaM-KI LiKb and XCaM-KIa, respectively (Fig. 1). To test whether XCaM-KI regulates CaM-KK on Thr181 or Thr178, we constructed mutants with a Thr-to-Asp substitution at the putative CaM-KK phosphorylation site. The mutants were named XCaM-KI LiKb T181D or XCaM-KIa T178D. The substitution at the phosphorylation site was predicted to mimic phosphorylation, and indeed, XCaM-KI LiKb T181D and XCaM-KIa T178D both showed increased kinase activities compared with that of the wild-type, which is consistent with previously reported findings in mammalian CaM-KI and CaM-KIV (Tokumitsu et al., 1994; Haribabu et al., 1995). These results suggest that XCaM-KI LiKb and XCaMKIa were regulated by an upstream kinase, probably X. laevis CaM-KK (Wayman et al., 2000). 3.4. Constitutively active XCaM-KI inhibits cell cleavage
Fig. 3. CaM binding activities and kinase activities of recombinant XCaM-KIs. (a) Purified GST-fusion XCaM-KIs were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R250, or blotted onto a PVDF membrane (Ca2q or EDTA). The blots were reacted with a biotinylatedCaM in the presence of either 1 mM CaCl2 (Ca2q) or 1 mM EDTA (EDTA). GST-XCaM-KI LiKß wild-type and GSTXCaM-KI LiKb (1–297) were indicate as WT and 1–297, respectively. GST-XCaM-KIa wild-type and GST-XCaM-KIa (1–295) were indicate as WT and 1–295, respectively. Molecular mass markers are indicated on the left (kDa). Both wildtype XCaM-KI isoforms were capable of binding to CaM, whereas deletion mutants were incapable of CaM binding in the presence of calcium; (b) Kinase activities of both GSTfusion XCaM-KI isoforms were measured at 30 8C for 15 min. Values are mean"S.E.M. of triplicate experiments, each done in duplicate.
human CaM-KI (Haribabu et al., 1995; Verploegen et al., 2000), confirming that these two XCaM-KI isoforms are regulated by Ca2q yCaM and that
In order to examine the role of CaM-KI in X. laevis early development, we injected various mutants of XCaM-KI mRNA into X. laevis embryos. Both constitutively active mutants of XCaMKI, XCaM-KI LiKb (1–297) and XCaM-KIa (1–295), inhibited cell cleavage (Fig. 4a, constitutively active mutant). As expected from the homology with human CaM-KI (Haribabu et al., 1995), replacement of the essential lysine at the ATP-binding site to a glutamic acid (Lysine 53 for XCaM-KI LiKb, or Lysine 50 for XCaM-KIa) resulted in inactive enzymes in vitro (data not shown). Injection of this kinase negative mutant (Fig. 4a, kinase negative mutant: XCaM-KI LiKb w1–297, K53Ex or XCaM-KIa w1–295, K50Ex) did not show cell cleavage arrest, thereby confirming that cell cleavage arrest was caused specifically by the activated kinase activities of XCaM-KI. (The cleavage arrest observed here is not likely through transcription, since major zygotic transcription starts after mid-blastula transition at approximately stage 9.)
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that both CaM-KI isoforms are cytosolic proteins (Soderling and Stull, 2001). Both constitutively active mutants of XCaM-KI (XCaM-KI LiKb w1– 297x or XCaM-KIa w1–295x) caused abnormal morphology when transfected into Hela cells (Fig. 4b, middle panel, constitutively active mutant). As kinase negative mutants (XCaM-KI LiKb w1–297, K53Ex or XCaM-KIa w1–295, K50Ex) gave normal cell morphology in transfected cells (Fig. 4b, bottom panel, kinase negative mutant), this indicates that the cell abnormality observed was caused specifically by the constitutive kinase activity of XCaM-KI. 4. Discussion
Fig. 4. The effects of XCaM-KI in X. laevis embryos and cultured cells. (a) Overexpression of constitutively active CaMKI inhibited cell cleavage in X. laevis embryos. RNA encoding XCaM-KI LiKb (left, 125 pg) or XCaM-KIa (right, 62.5 pg) was injected into a single blastomere at 2-cell stage embryos. Injected embryos were cultured until stage 8. Constitutively active mutant, top panels; kinase negative mutant, bottom panels; (b) Subcellular localization and effects of XCaM-KI isoforms in Hela cells. Hela cells were transiently transfected in 6-well plates with 1 mg of pCS2q plasmids expressing Myctagged wild-types or mutants XCaM-KI isoforms. Wild-type XCaM-KI LiKb or XCaM-KIa (top, wild-type); XCaM-KI LiKb (1–297) or XCaM-KIa (1–295) (middle, constitutively active mutant); XCaM-KI LiKb (1–297, K53E) or XCaMKIa (1–295, K50E) (bottom, kinase negative mutant). After 24 h, the transfected cells were fixed, and immunofluorostained with an anti-Myc monoclonal antibody (9E10). Constitutively active mutants of both XCaM-KI isoforms caused abnormal morphology in Hela cells.
We next examined the subcellular localization of XCaM-KI isoforms and their functions in cultured cells. Both wild-type XCaM-KI LiKb and wild-type XCaM-KIa localized mainly in the cytoplasm (Fig. 4b, top panel, wild-type), confirming
We have shown that at least two different isoforms of CaM-KI are expressed in X. laevis early embryogenesis. These have been designated XCaM-KI LiKb and XCaM-KIa. When the cell cleavage progresses in X. laevis embryos, a transient rise of wCa2qxi is observed along the furrow (Muto et al., 1996). While it is known that cleavage furrow formation requires IP3 receptormediated Ca2q signaling (Mitsuyama et al., 1999), the role of Ca2q and its downstream targets are unclear. But we found that XCaM-KIa protein expressed throughout early cleavage stages (Fig. 2a), and it was activated by Ca2q yCaM (Fig. 3b). Moreover, overexpression of a constitutively active mutant of CaM-KI isoforms caused cell cleavage arrest in X. laevis embryos (Fig. 4a), while overexpression of either constitutively active CaM-KII or CaM-KIV, which are other multi-functional CaM-K, does not inhibit cell cleavage in X. laevis ¨ et al., 2000; Wayman et al., 2000). embryos (Kuhl Therefore, XCaM-KIa may have a role in cell cleavage progression. The cleavage arrest phenotype is also observed in the activation of the MAPK signaling pathway in X. laevis embryos (Haccard et al., 1993; Kosako et al., 1994; Bhatt and Ferrell, 1999; Gross et al., 1999). Moreover, the MAPK pathway is activated not only by the calcium ionophore (Franklin et al., 2000) but also by CaM-K (Enslen et al., 1996; Verploegen et al., 2000). We therefore tested the possibility of the activation of the MAPK pathway by XCaM-KI. However, neither XCaM-KI LiKb (1–297) nor XCaM-KIa (1–295) could activate MAPK in embryos, nor did they cause germinal vesicle break down in the X. laevis oocytes (T.S.
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unpublished results). Therefore, XCaM-KI is unlikely to activate the MAPK signaling pathway. We also studied whether CaM-KI had a role in microtubule dynamics, as has been shown for CaM-KIV. CaM-KIV regulates microtubule dynamics through phosphorylation oncoprotein 18 on Ser 16 (Marklund et al., 1994; Melander Gradin et al., 1997). Considering the substrate similarities between CaM-KI and -KIV, it is possible that XCaM-KI also regulates microtubule dynamics. Overexpression of constitutively active XCaM-KI mutants, however, had no effect on gross cellular distribution of tubulin protein in Hela cells (T.S. unpublished result), thereby showing that tubulin might not be the downstream substrate for XCaMKI. Muto and Mikoshiba have reported that a transient increase of Ca2q induces cell shape changes in fertilized eggs and that the contracted region is strongly stained with rhodamine-phalloidin (Muto and Mikoshiba, 1998). Taken together, our results suggest that cytoskeletal proteins, e.g. F-actin, may be candidate targets regulated by Ca2q through the action of CaM-KI. Of additional interest is that the CaM-KI LiK amino acid in humans and the XCaM-KI LiKb amino acid in X. laevis is almost the same, except for the C-terminal portion. The human homologue of CaM-KI LiKb (GenBank accession number AX167585) shows very high similarities with human CaM-KI LiK, which is 97.5% identical at the amino acid level. It is possible these two isoforms are alternatively spliced variants, as are CaM-KI b1 and b2 (Naito et al., 1997). At present, X. laevis CaM-KI LiK and human CaMKI LiKb have been not reported; however, human CaM-KI LiK highly expresses in polymorphonuclear leukocytes (Verploegen et al., 2000), while XCaM-KI LiKb highly expressed in brain and testis (Fig. 2b). These differences suggest that the C-terminal sequence of CaM-KI LiK may have an important role for isoform-specific characteristics or tissue distribution. Future studies will examine the endogenous substrate of XCaM-KI LiKb, and its functional consequences. Acknowledgments We thank Drs T. Chatila, D. Melton and D. Turner, for the reagents. We thank Drs A. Mizutani and H. Tokumitsu for their critical discussion of the manuscript, and Y. Takeyama for technical
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