Organization and evolution of multifunctional Ca ... - people.vcu.edu

Gene 322 (2003) 17 – 31 www.elsevier.com/locate/gene

Review

Organization and evolution of multifunctional Ca2+/CaM-dependent protein kinase genes Robert M. Tombes a,b,c,d,*, M. Omar Faison a,c, J.M. Turbeville a,d a

Department of Biology, Virginia Commonwealth University, Richmond, VA 23284-2012, USA b Department of Biochemistry, Virginia Commonwealth University, Richmond, VA, USA c Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA d Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA, USA Received 15 July 2003; received in revised form 8 August 2003; accepted 26 August 2003 Received by A.J. van Wijnen

Abstract The ‘‘multi-functional’’ Ca2 + and calmodulin-dependent protein kinase, type II (CaMK-II) is an evolutionarily conserved protein. It has been found as a single gene in the horseshoe crab, marine sponge, sea urchin, nematode, and fruit fly, whereas most vertebrates possess four genes (a, h, g, and y). Species from fruit flies to humans encode alternative splice variants which are differentially targeted to phosphorylate diverse downstream targets of Ca2 + signaling. By comparing known CaMK-II protein and nucleotide sequences, we have now provided evidence for the evolutionary relatedness of CaMK-IIs. Parsimony analyses unambiguously indicate that the four vertebrate CaMK-II genes arose via repeated duplications. Nucleotide phylogenies show consistent but moderate support for the placement of the vertebrate y CaMK-II as the earliest diverging vertebrate gene. y CaMK-II is the only gene with both central and C-terminal variable domains and has three to four times more intronic sequence than the other three genes. h and g CaMK-II genes show strong sequence similarity and have comparable exon and intron organization and utilization. a CaMK-II is absent from amphibians (Xenopus laevis) and has the most restricted tissue specificity in mammals, whereas h, g, and y CaMK-IIs are expressed in most tissues. All 38 known mammalian CaMK-II splice variants were compiled with their tissue specificity and exon usage. Some of these variants use alternative 5Vand 3Vdonors within a single exon as well as alternative promoters. These findings serve as an important benchmark for future phylogenetic, developmental, or biochemical studies on this important, conserved, and highly regulated gene family. D 2003 Elsevier B.V. All rights reserved.

1. Introduction CaMK-II, the multifunctional Ca2 + and calmodulindependent serine/threonine protein kinase, is encoded by no more than four genes (a, h, g, and y) in diverse eukaryotes (Tobimatsu and Fujisawa, 1989; Karls et al.,

Abbreviations: CaMK-II, Ca2+ and calmodulin-dependent protein kinase, type II; CaM, calmodulin; NCBI, National Center for Biotechnology Information; EMBL, European Molecular Biology Laboratory; TBR, tree bisection reconnection; PIC, parsimony informative characters; MPtrees, most parsimonious trees; TL, tree length; CI, consistency index; RI, retention index. * Corresponding author. Virginia Commonwealth University, 1000 West Cary Street, Richmond, VA 23284-2012, USA. Tel.: +1-804-8270141; fax: +1-804-828-0503. E-mail address: [email protected] (R.M. Tombes). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.08.023

1992; Nghiem et al., 1993; Schworer et al., 1993). CaMKII phosphorylates substrates important in transcription, secretion, ion channel regulation and morphogenesis (Schulman et al., 1992; Nairn and Picciotto, 1994; Braun and Schulman, 1995; Soderling et al., 2001; Hudmon and Schulman, 2002a,b). CaMK-II monomers form oligomers of up to 12 subunits (Kolb et al., 1998). Each monomer is composed of catalytic, variable and oligomerization (also known as association) domains. The amino-terminal 315amino-acid catalytic domain includes a CaM-binding site, which is part of the autoinhibitory arm (Schulman et al., 1992). The central variable domain (30 –100 amino acids) is subject to alternative splicing, leading to the remarkable diversity of this kinase family. The C-terminal 135-aminoacid association domain brings catalytic heads into proximity through oligomerization. CaMK-II is distinguished from other CaM kinases by its ability to oligomerize and

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R.M. Tombes et al. / Gene 322 (2003) 17–31

to autophosphorylate at Thr287 (Thr286 in a CaMK-II). Phosphorylation of Thr287 relieves the enzyme from its Ca2 +/CaM dependence. CaMK-IIs from invertebrates also have been shown to form oligomers and to autophosphorylate (Baitinger et al., 1990; Tombes and Peppers, 1995; GuptaRoy and Griffith, 1996). The concerted influences of CaM binding, autophosphorylation, and oligomerization are responsible for the unique and conserved ability of CaMK-IIs to interpret frequency- and amplitude-modulated Ca2 + signals (Hanson et al., 1994; Dosemeci and Albers, 1996; De Koninck and Schulman, 1998). CaMK-II diversity in all species is achieved through alternative promoter and/or exon usage (Griffith and Greenspan, 1993; Mayer et al., 1993, 1994, 1995; Schworer et al., 1993; Edman and Schulman, 1994; Ramirez et al., 1997; Tombes and Krystal, 1997; Bayer et al., 1998, 1999; Hoch et al., 1998, 2000; Takeuchi and Fujisawa, 1998; Donai et al., 2000b; Mima et al., 2001; Hudmon and Schulman, 2002a). As many as seven alternative variable domain exons are used in different combinations to yield over three dozen unique isozymes (Tombes and Krystal, 1997; Bayer et al., 1998; Takeuchi et al., 2000a). These alternative variable domains and gene-specific targeting domains provide subcellular targeting to locations such as the nucleus, the plasma membrane, the actin cytoskeleton, and post-synaptic densities (PSD) (Srinivasan et al., 1994; Urquidi and Ashcroft, 1995; Heist et al., 1998; Shen and Meyer, 1999; Takeuchi et al., 2000a; Caran et al., 2001). An understanding of the expression patterns and roles of CaMK-IIs provides an important context for examining their phylogenetic relationships. Drosophila melanogaster has a single CaMK-II gene, from which at least five variants are expressed that are important in development and behavior (Griffith and Greenspan, 1993; Ohsako et al., 1993; Takamatsu et al., 1994; VanBerkum and Goodman, 1995; Broughton et al., 1996; GuptaRoy et al., 1996; GuptaRoy and Griffith, 1996; VanBerkum, 1996; Kahn and Matsumoto, 1997; Koh et al., 1999; Beumer et al., 2002). The single CaMK-II gene in the nematode Caenorhabditis elegans (unc-43) has been implicated in diverse behavioral phenomena (Reiner et al., 1999; Troemel et al., 1999; Wang and Wadsworth, 2002). Neuronal roles of vertebrate CaMK-IIs, in particular a CaMK-II, have been well established (Hudmon and Schulman, 2002a). Interestingly, Xenopus laevis expresses splice variants from only three (h, g, and y) CaMK-II genes (Stevens et al., 2001). CaMK-II has been implicated in cell cycle control during M-phase of early embryos (Lorca et al., 1993; Morin et al., 1994; Stevens et al., 1999, 2001), in centrosome duplication in Xenopus oocytes (Matsumoto and Maller, 2002), M-phase progression in sea urchins (Baitinger et al., 1990), and in the limitation of axonal and dendritic elaboration in the amphibian brain (Zou and Cline, 1996, 1999; Wu and Cline, 1998). Even in the sponge, Suberites domuncula, a basal metazoan, which lacks a

nervous system, CaMK-II expression is regulated (Krasko et al., 1999). In a previous study, we analyzed mammalian CaMK-II cDNA sequences and developed a model of CaMK-II exon structure and utilization (Tombes and Krystal, 1997). We have now analyzed the relationships of all available invertebrate and vertebrate CaMK-II coding sequences. We have analyzed the intron and exon structure of the human CaMK-II genes and compiled all known mammalian splice variants. Our findings clearly indicate that vertebrate CaMK-IIs evolved via duplication of a single ancestral CaMK-II gene, resulting in the three Xenopus genes and four genes in higher vertebrates. These phylogenetic studies provide a necessary context for future analyses of the function or expression of any CaMK-II variant.

Table 1 The number of known CaMK-II genes for the indicated species is listed along with GenBank accession numbers for protein and nucleotide sequences used in this study Species

Number of genes

Protein locus number

nt Locus

Sponge (Suberites domuncula) Nematode (Caenorhabditis elegans) Sea urchin (Strongylocentrotus purpuratus) Horseshoe Crab (Limulus polyphemus) Fruit Fly (Drosophila melanogaster)

1

CAB59634

SDO19007

1

AAF63320

AF233262.1

1

AY359469

AY359469

1

AAB40712

LPU49428

1

NP_524635

NM_079896.1

Vertebrates Frog (Xenopus laevis) Chicken (Gallus gallus) Rabbit (Oryctolagus cunuculus) Mouse (Mus musculus)

3 (h, g, y)

h: AAA81938 g: AAG17555 y: AAG17554 2 reported (a, h) a: AAC98390 h: AAC79460 2 reported (g, y) g: BAA28869 y: BAA28870

U06636.1 AF233630 AF233629.1 AF109069 AF085249 D14905.1 D14906.1

4 (a, h, g, y)

X14836 NM_007595.1 AF395884.1 AK012702.1 J02942.1 NM_021739.1 S71571.1 NM_012519.1 AF145710.1 NM_001220.2 XM_044349.6 NM_001221.1

Rat (Rattus norvegicus)

4 (a, h, g, y)

Human (Homo sapiens)

4 (a, h, g, y)

a: CAA32946 h: P28652 g: AAK84142 y: BAB28422 a: AAA41870 h: S68470 g: AAB30671 y: A34366 a: AAD30558 h: NP_001211 g: B46619 y: Q13557


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Fig. 1. Catalytic domain alignment. Amino acid sequences in the catalytic domain were aligned by clustal analysis using Gene Jockey-II software (BioSoft, Cambridge UK) or by using ClustalW as implemented in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Identical residues among all 20 sequences are indicated by dots and similarities by vertical lines. Known exon boundaries are outlined in alternating light and gray boxes. Exons were marked as beginning or ending if they contained 2/3 of a codon. Vertical boxes indicate gene-specific residues. Alignments have been deposited in the EMBL align database.

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2. CaMK-II sequences All available CaMK-IIs were identified from public databases across taxa by the presence of (a) an oligomerization domain, (b) a CaM binding domain, (c) the stimulatory autophosphorylation site (Thr287) in the CaM-binding domain, and (d) the inhibitory phosphorylation site (Thr306). These sequences were complete, except for urchin and crab. Both biochemically and by this analysis, neither yeast (Saccharomyces cerevisiae) nor plant (Arabidopsis thaliana) CaM kinase sequences (Hong et al., 1996) are included, as they lack these features. A recent analysis of CaM-binding proteins in Arabidopsis also concludes that CaMK-IIs are absent (Reddy et al., 2002). A compilation of the species where CaMK-IIs have been identified, based on the above

properties, is shown with their known CaMK-II genes, and the NCBI protein or gene loci (Table 1). Those species include the marine sponge (S. domuncula) (Krasko et al., 1999), the nematode (C. elegans) (Reiner et al., 1999), the sea urchin (Strongylocentrotus purpuratus) (Baitinger et al., 1990; Tombes and Peppers, 1995), the horseshoe crab (Limulus polyphemus) (Calman et al., 1996), insects (D. melanogaster) (Ohsako et al., 1993), the African clawed toad (X. laevis) (Stevens et al., 2001), chicken (Gallus gallus) (Li et al., 1998), rabbit (Oryctolagus cunuculus) (Takeuchi and Fujisawa, 1998), mouse (Mus musculus) (Hanley et al., 1989; Karls et al., 1992), and human. Rat (Rattus norvegicus) CaMK-IIs have been thoroughly studied (Bennett and Kennedy, 1987; Lin et al., 1987; Tobimatsu and Fujisawa, 1989; Schworer et al., 1993; Urquidi and Ashcroft, 1995;

Fig. 2. Variable and association domain alignment. Amino acid sequences in the C-terminal half were aligned by clustal analysis. Sequences commenced in the variable domain and included only the conserved linker exons (II and VII), followed by the three association domain exons and the alternative C-terminal exon in y CaMK-IIs. Identical sequences among all 20 sequences are indicated by dots. Sequence similarities are indicated by vertical lines. Exons are outlined by alternating light and dark boxes. Vertical boxes indicate gene-specific residues.


Zhou et al., 1995) and their accession numbers listed in Table 1. However, they are not included in further analyses because of their near-perfect identity (>99%) with mouse sequences. The sea urchin CaMK-II sequence was determined from partial sea urchin CaMK-II clones which were obtained from a sea urchin Egt10 library of S. purpuratus ovarian cDNA (courtesy of Gary M. Wessel, Brown Univ.). This library was screened with a probe prepared from the CaMK-II catalytic domain.

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Table 2 Number of parsimony informative characters and tree statistics for nucleotide data Value

All positions

First and second

Third

PIC MPtrees TL CI RI

741 2 3299 0.43 0.50

275 9 712 0.57 0.68

466 3 2538 0.40 0.45

PIC, parsimony informative characters; MPtrees, most parsimonious trees; TL, tree length; CI, consistency index; RI, retention index.

3. Phylogenetic analyses Amino acid and nucleotide sequences were aligned separately. Protein sequences of 20 different CaMK-IIs from vertebrates (human, mouse, rabbit, chicken, frog) and invertebrates (fruit fly, nematode, horseshoe crab, marine sponge, and sea urchin) were aligned by clustal analysis (Wilbur and Lipman, 1983), omitting alternative sequences from the central region (Figs. 1 and 2). Sequences are shown aligned with dots indicating identity and vertical lines indicating conservative replacements among all 20 sequences. Horizontal boxes indicate known exon boundaries and vertical boxes mark gene-specific sequences. Pairwise similarity ranged from 48% to 100%. Of the 509 amino acid positions, 151 were parsimony informative. Parsimony analysis of these data for all 20 sequences resulted in two equally

parsimonious trees (length = 469; CI = 0.78; RI = 0.81). The bootstrap majority-rule consensus tree is shown in Fig. 3A. The four vertebrate CaMK-II genes are strongly supported as distinct clades with bootstrap values of 99% (y) or 100% (a, h and g). Within the vertebrate lineage, the h genes are most closely related to the g genes (89%), whereas the y genes are most closely linked to the a genes, although with weaker bootstrap support (58%). For all phylogenies in Fig. 3, bootstrap values are indicated above the nodes and represent the percentage of trees which contain the clade descending from that node in 2000 replicates (see Swofford et al., 1996 for further description of these analytical approaches). Nucleotide sequences were aligned by invoking the ‘toggle translation’ option of BioEdit (http://www.mbio.ncsu. edu/BioEdit/bioedit.html), which places gaps in the nucleo-

Fig. 3. Gene phylogenies inferred by parsimony analysis of entire coding sequence. Phylogenies were reconstructed using unweighted parsimony analysis as implemented in PAUP*4.0 (Swofford, 2002). For analyses of the nucleotide data set, a heuristic search was used with 2000 random addition replicates and TBR (tree bisection reconnection) branch swapping, whereas for the amino acid data set, a branch and bound search was performed. Branch support was assessed using bootstrap analysis with 2000 pseudoreplicates and either a full heuristic search (nucleotide data) or a branch and bound search (amino acid data). For all phylogenies, numbers above the nodes correspond to the percentage of trees containing the clade descending from that node in the 2000 replicates. All tree statistics were determined excluding uninformative characters. Sequence gaps were treated as missing data in all analyses. Suberites (Porifera) was selected as the outgroup for all phylogenetic analyses because both morphological and molecular data suggest that the Porifera is the basal metazoan taxon. (A) Bootstrap majority-rule consensus tree found by analyzing amino acid sequences. (B) Bootstrap majority-rule consensus tree inferred from analysis of all informative nucleotide positions. (C) Bootstrap majority-rule consensus tree estimated from analysis of first and second codon positions.

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tide sequences corresponding to those in the amino acid sequence alignment. As with amino acids, parsimony analysis of nucleotides strongly supports the existence of four distinct vertebrate CaMK-II genes. The y and a clades are supported strongly (100% bootstrap values each), whereas the g clade received less support (82%). A h group, exclusive of the Xenopus gene, is also strongly supported (98%). This analysis suggests that the Xenopus h gene is the sister group to a clade comprised of the a gene group plus a group consisting of the other vertebrate h genes (Fig. 3B). The vertebrate y gene group is at the base of the vertebrate gene

phylogeny (80% bootstrap value), suggesting that this gene diverged first. Nucleotide data sets have inherently more informative characters than corresponding amino acid data sets, but the more variable third position may exhibit ‘‘noise,’’ which could lead to artifactual phylogenetic groupings (Swofford et al., 1996). Uncorrected pairwise divergence values were therefore determined at each codon position. Those data show that there is greater variation for the third (0.11– 0.69) than for the first (0.006 – 0.39) and second positions (0– 0.28). The third position also exhibits more homoplasy than

Fig. 4. Human CaMK-II gene structure and variable domain exon options. Boxes represent exons connected by lines across introns which are drawn using the scale indicated for each gene. Alternative 5Vor 3Voptions are indicated. Catalytic domain exons are numbered 1 – 12, variable domain exons are numbered with roman numerals I through X, and association domain exons are consecutively numbered with the last (large) exon representing the 3V untranslated region. Promoter positions (arrows) are approximate. Catalytic domain 1 also contains a 5Vuntranslated region.


the first and second positions, as indicated by the lower consistency index (CI) and retention index (RI) values (Table 2). Nucleotide data were therefore reanalyzed by parsimony analysis excluding position 3 (Fig. 3C). Unlike the full nucleotide data set, this analysis shows strong support (98%) for the inclusion of the Xenopus h gene within the h clade. This analysis also more strongly supports monophyly of the other three genes. Finally, as observed with the full nucleotide data set, this analysis shows the y clade as the earliest to diverge, although bootstrap support is weak (57%). Very similar consensus trees were observed when only the catalytic domain sequences were used, but association domain sequences alone were unable to resolve any gene hierarchy (not shown). The total nucleotide data set was also analyzed using maximum likelihood analysis, an alternative phylogenetic tool. The best-fitting model (GTR + G + I) for the data was determined using MODELTEST (Posada and Crandall, 1998). Two heuristic search strategies were employed to find optimal trees. The first implemented the ‘‘likelihood ratchet’’ (Vos, 2003) with 118 iterations. In the second analysis, equally parsimonious trees generated from the complete nucleotide data set were input as starting trees and tree bisection reconnection (TBR) branch swapping allowed until completion. The topology of the maximum likelihood tree was identical to the parsimony bootstrap-consensus tree for first and second nucleotide positions (Fig. 3C). In summary, both nucleotide and amino acid sequence phylogenies unambiguously support the origin of the vertebrate CaMK-II genes via duplication of an ancestral form of the gene. With the exception of the total nucleotide data set, which excludes the frog h from a group comprised of the other h genes, all analyses support four vertebrate CaMK-II gene groups. Any topological discrepancies may be attributed to misleading signal at the third codon position, as all h sequences are linked strongly when the third position is excluded (Fig. 3C). Analyses of the complete nucleotide sequences and the sequences excluding the third codon position find moderate support for the placement of the vertebrate y CaMK-II as the earliest diverging vertebrate gene. Analysis of the amino acid sequences, however, does not place any one CaMK-II gene as the most ancestral. Amino acid and first and second codon position analyses support a close relationship between the h and g genes (Fig. 3A and C), however, their placement relative to a and y clades is not strongly supported even when outgroup selection was varied or when catalytic and association domain sequences were analyzed separately (not shown). Thus, the precise relationships of the vertebrate CaMK-II gene groups cannot be unequivocally established with these data. The relationships among the invertebrate (fruit fly, nematode, horseshoe crab, marine sponge and sea urchin) genes were also not resolved with these data. Possible reasons for the observed topological incongruence include differing information content in amino acid versus nucleotide sequences, disparate rates of sequence evolution, and insufficient sam-

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pling of CaMK-II genes among species. Additional data, particularly from ‘‘lower vertebrates’’ (e.g., fishes) and other deuterostomes (Hemichordata, Urochordata, Cephalochordata, Echinodermata), will be necessary to definitively establish vertebrate CaMK-II gene evolution.

4. Human gene structure CaMK-II gene relatedness can also be evaluated by examination of genomic organization. The four human CaMK-II genes are shown with chromosome location and relative exon size (Fig. 4). Exon placement and total gene size were determined by comparing mammalian CaMK-II cDNAs to human genomic sequences (NCBI). Automated methods to identify intron/exon junctions developed by NCBI have since confirmed our findings. Exon and intron Table 3 Human CaMK-II exon size and properties a

g

y

Function

Catalytic domain: 1 62 65 65 2 95 95 95 3 60 60 60 4 55 55 55 5 66 66 66 6 73 73 73 7 103 103 103

65 95 60 55 66 73 103

8 84 9 95 10 123 11 84 12 43

84 95 123 84 43

KD-I: GxGxxS-ribose anchor-bridges KD-II: catalytic K43 KD-III: E61, bridge to K43 KD-IV: not known KD-V: not known KD-VI: Ser/Thr specific residues KD-VII: His, Arg coordination of g-phos KD-VIII, IX: activation loop KD-X: not known KD-XI: CaMK-II specific CaM-binding, autophos. T287 Inhibitory autophosphorylation: T306

h

84 95 123 84 43

Variable domain: 13 – 75 14 41* 41* 15 33 – 16 – 72 17 – 45

84 95 123 84 43

63 44 33 69** 45

– 38* 33 60 42

18 19

49 –

49 –

49 114*

49 –

20 21 22

– – –

114 129 129

– – –

– – –

Association domain: 23 76 76 76 24 95 95 95 25 230 230 230 26 – – –

76 95 230 64

Variable Domain I: CaM Affinity reg. Variable Domain II: Linker 1 (aKAP) Variable Domain III: Nuclear targeting Variable Domain IV/V: not known Variable Domain VI: Nuclear targeting inhibitor Variable Domain VII: Linker 2 Variable Domain VIII/IX: g-specific exon Variable Domain Xa: h-specific exon 1 Variable Domain Xb: h-specific exon 2 Variable Domain Xc: h-specific exon 3

Oligomerization Oligomerization Oligomerization Oligomerization

domain domain domain domain

I II III IV: y-specific

Human CaMK-II exon structure is listed for each gene with nucleotide (nt) length and a description of the encoded domain. The size of exon 1 is exclusive of the 5Vuntranslated region. Exons encoding 3Vuntranslated regions are also excluded from this summary. Catalytic and Association Domain exon boundaries are summarized in Figs. 1 and 2. * Alternative splice acceptors (5Vend of exon). ** Alternative splice donors (3Vend of exon).

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sizes have also been determined in mouse a, h (Karls et al., 1992) and y and in rat a (Nishioka et al., 1996) and h (Donai et al., 2001) and are similar to the findings in human genes. Human a CaMK-II covers 70 kb on chromosome 5 and encodes 19 exons with two different transcriptional promoters, one of which is internal, which leads to the production of the aKAP protein. Human h CaMK-II spans 108 kb on chromosome 7 and encodes 24 exons. Human g CaMK-II covers 62 kb on chromosome 10 (Li et al., 1994) and encodes 23 exons. Human y CaMK-II spans 309 kb on chromosome 4 and encodes 22 exons. y CaMK-II exons occur, on average, every 14 kb, in contrast to the 4-kb average for a and h CaMK-IIs. When mouse and human a and y CaMK-II genes were compared, human introns were an average of 15% longer than mouse CaMK-II introns, but exon lengths were identical. Exon sizes were summarized for all four human genes (Table 3). There are 12 highly conserved exons in the catalytic domain; 10 different exons distributed among all four genes in the variable domain (Tombes and Krystal, 1997; Hudmon and Schulman, 2002a) and three moderately conserved exons encoding the association domain. 4.1. Catalytic domain structure Among human a, h, g, and y CaMK-IIs, the sizes of the first 12 exons are highly conserved (Table 3). Except for exon 1 in which a CaMK-II has one less codon, the other 11 exons are identically sized between genes and have similar

acceptor and donor sequences (not shown). The first eight exons correspond to the eight functional kinase domains (Hanks et al., 1988; Hanks and Quinn, 1991), such as the ribose anchor (catalytic domain 1, exon 1) and the activation loop (domain 8, exon 8), an observation also made in the rat a (Nishioka et al., 1996) and rat h CaMK-II gene (Donai et al., 2001). The CaM binding and stimulatory autophosphorylation (T287) sites are encoded by exon 11. Exon 12 contains the inhibitory T306 autophosphorylation residue (Patton et al., 1990). The most conserved exons are the eighth and ninth with 85 – 95% similarity or identity between all genes. 4.2. Variable domain structure The variable domain in mammalian CaMK-II genes has 10 separate exons (Table 3). Each human gene uses between 3 and 8 of these exons (Fig. 4). Most of these exons were previously proposed (Tombes and Krystal, 1997; Hudmon and Schulman, 2002a). The amino acid sequences of human variable exons I – VII (Singer et al., 1997; Tombes and Krystal, 1997; Takeuchi and Fujisawa, 1998) are summarized here (Table 4). cDNAs cloned from X. laevis suggest the presence of an additional exon at the beginning of the variable domain (Stevens et al., 2001), but this is absent from mammalian genes. Variable domains were originally numbered I through X and their gene-specific sequence listed (Tombes and Krystal, 1997). This numbering system was adopted and augmented (Stevens et al., 2001; Hudmon

Table 4 Human variable domain protein sequence properties and similarities h–I h–I 100 g–I 43 a – II 14 h – II 23 g – II 20 y – II 15 a – III 18 g – III 18 y – III 18 h – IV/V 21 g – IV/V 17 y – IV/V 20 h – VI 20 g – VI 20 y – VI 21 a – VII 19 h – VII 19 g – VII 25 y – VII 29

g – I a – II h – II g – II y – II a – III g – III y – III h – IV/V g – IV/V y – IV/V h – VI g – VI y – VI a – VII h – VII g – VII y – VII 100 14 100 23 64 13 57 23 46 27 27 27 27 27 27 24 14 24 21 15 21 13 21 13 21 29 21 12 14 19 14 12 14 12 14

100 78 62 18 18 18 23 23 31 23 23 23 15 15 15 23

100 54 27 27 27 20 20 13 13 13 14 20 20 20 27

100 18 100 18 91 18 91 23 18 23 18 15 18 23 9 23 18 31 9 8 27 15 27 15 18 15 18

100 82 18 18 18 9 18 9 27 28 18 18

100 18 27 18 9 9 9 27 27 18 18

100 22 35 27 27 29 19 19 19 18

100 30 20 12 14 19 19 12 12

100 20 12 21 12 12 12 12

100 80 71 20 13 13 20

100 57 20 13 20 20

100 29 21 21 29

100 81 69 81

100 62 75

100 75

100

Human CaMK-II variable domain protein sequences of varying size were aligned by clustal analysis. Scores less than 25 indicate little similarity and those higher than 25 are in bold. Human variable domain sequences: h – I, VGRQTTAPATMSTAASGTTMGLVEQ; g – I, VGRQSSAPASPAASAAGLAGQ; a – II, GGKSGGNKKSDGVK; h – II, AAKSLLNKKADGVK; g – II, AAKSLLNKKSDGGVK; y – II, AAKSLLKKPDGVK; a – III, KRKSSSSVQLM; g – III, KRKSSSSVHLM; y – III, KRKSSSSVQMM; h – IV/V, PQTNSTKNSAAATSPKGTLPPAAL; g – IV/V, PQSNNKNSLVSPAQEPAPLQTAM; y – IV/V, INNKANVVTSPKENIPTPAL; h – VI, EPQATVIHNPVDGIK; g – VI, EPQTTVVHNATDGIK; y – VI, EPQTTVIHNPDGNK; a – VII, ESSESTNTTIEDEDTK; h – VII, ESSDSANTTIEDEDAK; g – VII, GSTESCNTTTEDEDLK; y – VII, ESTESSNTTIEDEDVK.


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Table 5 Tissue specificity of variable domain utilization

All known mammalian CaMK-II isozymes are listed with current and alternative nomenclature, variable domain exon usage and tissue distribution. Exons are described in text and in Tables 3 and 4. Predicted Mr are listed.

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and Schulman, 2002a) and has been retained here, even though in two separate cases, separate domains are encoded by a single exon using alternative 5Vor 3Vsplice sites (Fig. 4). Alternative usage of variable domains is summarized in Fig. 4 and Table 5. A similarity comparison was conducted between variable domains I through VII to search for duplicated domains, such as with the triplicated h-specific domains Xa, Xb, and Xc (Urquidi and Ashcroft, 1995), and thus provide evidence for evolutionary relationships among variable domains. The results of this clustal analysis are shown in Table 4. Similarity values above 25% are highlighted in bold and not only support the groupings of similarly numbered variable domains between genes (46 –91%), but also between different variable domains. For example, domain y II shows 31% similarity to y VI and y IV/V shows 31% similarity to h II. Variable domain I is found only in the h and g genes and encodes either 21 or 25 amino acids. This domain has been shown to influence the binding affinity for CaM (Brocke et al., 1999) and the ability of CaMK-IIs to autophosphorylate (Bayer et al., 2002). Variable domain II is not alternative; it is found in all isozymes from all genes. It was first described as a ‘‘linker’’ domain between the catalytic and association domains (Kanaseki et al., 1991). This domain varies in length and sequence between genes, encoding 13– 15 residues, but is conserved between genes (46 – 78% similarity). Nonetheless, in the a gene, domain II is encoded by an exon with an alternative splice acceptor (5Vend), which in response to an internal promoter gives rise to the aKAP gene product (Bayer et al., 1996, 1998; Sugai et al., 1996; Takeuchi and Fujisawa, 1997). We have denoted aKAP domain II by an asterisk, since it contains a 25-amino-acid hydrophobic leader sequence attached to domain II. The resulting aKAP protein is catalytically inactive, but can hetero-oligomerize and target active CaMK-II to membranes. Variable domain III encodes a highly conserved 11residue nuclear targeting sequence and is found in the a, g, and y, but not the h CaMK-II gene. Domain III can exert a dominant targeting role in hetero-oligomeric CaMK-II (Srinivasan et al., 1994). The proline-rich variable domain IV/V is encoded by a single exon in the h, g, and y genes. In the g gene, this exon utilizes an alternative donor site (3V termination) enabling domain IV to be expressed alone in certain g isozymes. In contrast, h and y CaMK-II isozymes always use the entire exon to encode a single IV/V domain as substantiated by the absence of potential 3V splice sites within this exon. All h isozymes, except h2, use this exon (Rochlitz et al., 2000). There is no function yet assigned to variable domain IV/V and it is the least conserved among genes. Variable domain VI, found in h, g, and y genes, has been implicated in the reversible inhibition of g CaMK-II nuclear targeting by domain III (Takeuchi et al., 2000a). Nuclear targeted a CaMK-IIs cannot be subjected to this inhibition

since domain VI is absent from the a gene. In contrast, the h gene has domain VI, but not domain III. In y CaMK-IIs, domain VI shows sequence similarity (PDGXKE) to domain II (Table 4), suggesting duplication of this exon (Johnson et al., 2000). Variable domain VII, like domain II, is found in all isozymes of all genes. Domains II and VII were therefore included in the sequence alignments and phylogenetic analysis (Figs. 1 –3). Domains II and VII are also referred to as segments B and C (Hudmon and Schulman, 2002a). Domain VII is not only conserved in usage, but in nucleotide length and amino acid sequence among all four genes. This domain is believed to form a second linker or tether sequence between functional domains. Clustal analysis indicates that this is the most highly conserved variable domain exon between genes (Table 4). Variable domain VIII/IX is a g-specific 114-nt exon (Tombes and Krystal, 1997). Much like exon IV/V, these two domains are encoded by a single exon, which is subject to an alternative acceptor site (alternative 5V donor) in the middle of the exon. There is no function yet determined for this variable domain. Variable domain X is triplicated to encode domains with SH3 binding potential (Urquidi and Ashcroft, 1995). The three Domain X exons (Xa, Xb, and Xc) are only found in the h CaMK-II gene, with no similarity to other genes. Xa is never expressed alone, Xa and Xb are expressed in isozyme h3, while all three are utilized in splice variant hM. 4.3. Association domain structure The association domain is encoded by three exons in all genes (Table 3). There are three hydrophilic domains proposed to be responsible for oligomerization (Kolb et al., 1998). The first two domains span the two junctions of these three exons, whereas the last domain is encoded within the third exon. At the C terminus of y CaMK-II, there is an alternative additional exon, which encodes a 21amino-acid tail with unknown function (Schworer et al., 1993; Hoch et al., 1998).

5. Tissue specificity of CaMK-II isozymes Finally, in order to examine whether function correlates with evolutionary relationships, we have compiled a list of all 38 known mammalian isozymes with their exon usage, predicted Mr, and tissue specificity (Table 5). Although it is possible that more isozymes will be found which utilize alternative combinations of these variable domains, it is unlikely that additional domains will be found. Each isozyme has been detected as cDNA and/or protein in human or rodent tissues. Many of these isozymes have been found by more than one group and often have multiple names as first described (Tombes and Krystal, 1997) and further elaborated (Hudmon and Schulman, 2002a). In general, no


hard rules for the tissue specificity of alternative splice variants in this gene family have been established. Only a few isozymes (encoded by the a gene) can be described as strictly tissue-specific. Rather, CaMK-II variants undergo a prolonged transition in expression from undifferentiated to differentiated tissues. The a CaMK-II gene encodes three variants. a and a33 (or aB) are only expressed in selected regions of the central nervous system after birth (Bayer et al., 1999). Full-length a CaMK-II is important in behavior and memory through its influence on long-term potentiation (LTP) at post-synaptic densities (PSD), but is not necessary for embryonic development (Silva et al., 1992; Chen et al., 1994; Mayford et al., 1996). It is specific to frontal cerebral cortex (Miller and Kennedy, 1985; Beaman-Hall et al., 1992; Takeuchi et al., 2002). Nuclear targeted aB is found only in the striatum and midbrain (Brocke et al., 1995), where it may influence BDNF expression (Takeuchi et al., 2000b). aKAP has only been detected in cardiac and skeletal muscle, where it oligomerizes with and targets active CaMK-II to the sarcoplasmic reticulum (Bayer et al., 1996, 1998; Sugai et al., 1996; Takeuchi and Fujisawa, 1997). h CaMK-II is encoded as seven splice variants, none of which possesses nuclear targeting sequences. h CaMK-II expression is strongest in the adult brain, but also occurs in skeletal muscle (Karls et al., 1992), small intestine (Tobimatsu and Fujisawa, 1989), and in endocrine tissues, such as the pancreas, adrenal, and pituitary (Urquidi and Ashcroft, 1995; Breen and Ashcroft, 1997; Rochlitz et al., 2000). Fulllength h CaMK-II is found throughout the brain, but is enriched in the cerebellum (McGuinness et al., 1985; Miller and Kennedy, 1985; Beaman-Hall et al., 1992; Takeuchi et al., 2002). Embryonic mouse brain expresses a spectrum of h isozymes, including hV, he, and heV, which are replaced by full-length h within 2 weeks of birth (Brocke et al., 1995; Urushihara and Yamauchi, 2001). Mouse h CaMK-II knockouts are embryonic lethal (Karls et al., 1992). h3 and hM, which are presumably targeted to the plasma membrane, are expressed in rodent endocrine and muscle tissue (Urquidi and Ashcroft, 1995; Breen and Ashcroft, 1997; Bayer et al., 1998, 2002; Gloyn and Ashcroft, 2001). hV (h1), he (h4) and h2 are all found at significant levels in the adrenal, pituitary and in pancreatic h cells (Rochlitz et al., 2000). Full-length h CaMK-II has been shown to interact with the actin cytoskeleton (Shen and Meyer, 1999). The g CaMK-II gene encodes 13 known splice variants. There is agreement on the nomenclature and structure of gA, gB, gC, gE, and gF (Kwiatkowski and McGill, 1995; Singer et al., 1997; Tombes and Krystal, 1997; Takeuchi and Fujisawa, 1998; Hudmon and Schulman, 2002b). There has been disagreement on the nomenclature of gD, gG, gH, and gI. In Table 5, we have adhered to the nomenclature summarized for gD, gG, and gH (Hudmon and Schulman, 2002b), but have also included the structures of g*D (Kwiatkowski and McGill, 1995), g*G, g*H, and gI (Takeuchi and Fujisawa, 1998). g CaMK-II mRNA is ubiquitous, but is

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highest in brain and skeletal muscle and lowest in liver, testis, lung, thymus, and kidney (Tobimatsu and Fujisawa, 1989; Bayer et al., 1999). gC is the most ubiquitous g isozyme. g CaMK-IIs are expressed at high levels in both gallbladder and a human gallbladder cholangiocarcinoma cell line (Kwiatkowski and McGill, 1995). T lymphocytes are a rich source of g isozymes (Nghiem et al., 1993). The nuclear targeted g CaMK-II variant, gA, has only been detected in the brain, where it is enriched in cortex, and in astrocytes (Vallano et al., 2000). Like gA, gAVcontains both variable domains III (nuclear targeting) and VI (inhibitor of nuclear targeting), but has only been detected in the striatum (Takeuchi et al., 2002). gH* also contains domains III and VI and was discovered in rabbit liver (Takeuchi and Fujisawa, 1998). gB and gE have been found in human islets (Breen and Ashcroft, 1997), while gG and gH were cloned from human mammary epithelial cells (Tombes and Krystal, 1997). g CaMK-II gene products have been attributed to many roles in the immune and endocrine systems (MacNicol et al., 1990; MacNicol and Schulman, 1992; Nghiem et al., 1993; Cui et al., 1994; Norling et al., 1994; Urquidi and Ashcroft, 1995; Breen and Ashcroft, 1997; Singer et al., 1997; Shen et al., 1998) as reflected in their ubiquitous expression. The y CaMK-II gene encodes 15 splice variants from two variable regions (central and C-terminal). y isozymes use both number and letter subscripts, as recently summarized (Hudmon and Schulman, 2002a). y CaMK-IIs are important in embryogenesis and in cardiac and neuronal morphogenesis (Scholz et al., 1988; Brocke et al., 1995; Solem et al., 1995; VanBerkum and Goodman, 1995; Masse´ and Kelly, 1997; Tombes and Krystal, 1997; Hoch et al., 1998, 2000; Bayer et al., 1999; Tombes et al., 1999; Donai et al., 2000a; Johnson et al., 2000; Takeuchi et al., 2000a). y CaMK-II mRNA is also expressed at high levels in differentiated tissue such as brain, skeletal, and cardiac muscle, with significant quantities in intestine and lung (Tobimatsu and Fujisawa, 1989; Bayer et al., 1999). Within the adult brain, y CaMK-II is most enriched in the cerebellum (Bayer et al., 1999). yA CaMK-II is a cytosolic variant enriched in the cerebellum, but also found in cortex and striatum (Vallano et al., 2000; Takeuchi et al., 2002). The nuclear targeted yB CaMK-II (y3) is enriched in adult cardiac and smooth muscle cells (Schworer et al., 1993), absent from myoblasts and myotubes (Hoch et al., 1998), but also present in the cerebellum, along with two other weakly expressed, but similar, nuclear targeted variants, y3.1 and y3.4 (Vallano et al., 2000). Isozymes containing the alternative C-terminal tail have been shown to be developmentally regulated during neurogenesis of P19 mouse embryonal carcinoma cells (Faison et al., 2002). Embryonic or tumor cell y CaMK-IIs are primarily non-nuclear, such as y2 (yC) and y6 (yG) (Bayer et al., 1999; Hagemann et al., 1999; Tombes et al., 1999; Hoch et al., 2000). y6, y7, y8, y9, and y10 CaMKIIs are all found in mouse heart (Hoch et al., 2000). y CaMK-IIs are not expressed in T lymphocytes (Nghiem et

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al., 1993), but have been found in endocrine tissues (Mohlig et al., 1997; Southam et al., 1999; Rochlitz et al., 2000). Two liver variants, y11 (yK) and y12 (yL), are similar to y2 and y6, except for single codon deletions via splicing (Takeuchi and Fujisawa, 1998).

6. Perspective As additional complete CaMK-II gene sequences are determined from other species, it will be important to include them in analyses similar to the present study. To help guide the classification of ambiguous sequences, approximately three dozen gene-specific amino acid residues were identified (Figs. 1 and 2, vertical boxes) throughout the catalytic and association domains. Although their function is unknown, these residues are diagnostic for each gene. Some examples include F55 and K67 in all invertebrate sequences except the sponge and H55 and L67 in every vertebrate CaMK-II. At position 264, all a CaMK-IIs encode alanine, h CaMK-IIs histidine, y CaMK-IIs serine or threonine, and g CaMK-IIs aspartic acid. The organization of CaMK-II genes reveals differences between the four mammalian genes. Human and rodent y CaMK-II genes are three- to fourfold larger than a, h, and g CaMK-II and have the greatest number of alternative splice variants. Unlike the other three genes, y CaMK-II variants are produced through the utilization of alternative exons in both the central variable region and at the C terminus. Within the central variable region, some of the strongest similarities occurred between h and g CaMK-IIs. For instance, only h and g CaMK-IIs contain variable domain I. Variable domains II and VI also show the most similarity between h and g CaMK-II sequences. These similarities are congruent with the protein and the first and second nucleotide phylogenies (Fig. 3), which suggests that h and g CaMK-II are most closely related. This analysis has also confirmed the structure and means by which exons are utilized in every one of the 38 known mammalian splice variants (Table 4). Although splicing was not directly evaluated, this analysis has uncovered examples of alternative 5V and 3V acceptor and donor sites within the same exon. The findings of this analysis confirm and extend the exon utilization model implicated from a previous analysis of cDNA sequences alone (Tombes and Krystal, 1997). All reported mammalian CaMK-II isozymes are listed here with their exon usage, predicted Mr, and tissues in which they have been found. Although additional isozymes may be found, this serves as an important and necessary consensus for isozyme nomenclature in this complicated gene family. Since CaMK-II can form mixed oligomers to create even further diversity in this family, it is important to have a clear understanding of which spectrum of isozymes is present in any one tissue. The absence of a CaMK-II from amphibians (Stevens et al., 1999, 2001) suggests that a CaMK-II may represent an

evolutionary novelty (derived trait) of birds and mammals. Furthermore, while h, g, and y CaMK-IIs are ubiquitous, a CaMK-II gene expression is the most specialized in mammals, being absolutely limited to the central nervous system, primarily the frontal cortex, and to muscle, as the truncated aKAP. CaMK-II is established as a well-conserved gene family present in species that have existed for 600 million years. CaMK-II is dependent on calmodulin (CaM) for activity; CaM is even more conserved, showing distribution in plants, yeast, and other species that lack CaMK-II. This analysis provides conclusive evidence for distinct evolution of the four vertebrate CaMK-IIs via multiple duplication of a single ancestral gene. Taken together, this analysis indicates that y CaMK-IIs are most ancestral, that h and g CaMK-IIs are related, and that a CaMK-II, which is the most functionally specialized, is unique to birds and mammals. These findings serve as a benchmark for future developmental and phylogenetic studies on CaMK-IIs and provide insight into the conservation of mechanisms for decoding Ca2 + signals.

Acknowledgements This work was supported by National Science Foundation grants 9904765 (RMT), 0089654 (JMT), an R. Clifton Brooks grant (RMT) and a Massey Cancer Center Training Grant (MOF). The authors are extremely grateful to Lenny Rebhun, Steve Ashcroft and Jim Roesser for their collaboration and interest in this project and to Mandy Itnyre for assistance with sequence analysis.

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