Studies of the Evolution of Creatine and Arginine Kinases in the Basal Metazoan Sponges

Creatine kinase (CK) and arginine kinase (AK) are members of a highly conserved family of phosphoryl transferase enzymes known as phosphagen kinases. CK and AK play a central role in cells that display high and variable rates of ATP turnover such as neurons, muscle fibers, spermatozoa, and transport epithelia. All CK isoforms exhibit targeting to specific tissues. In vertebrates CK exists as two dimeric, cytoplasmic isoforms, B (brain) and M (muscle) and two octameric, mitochondrial isoforms termed sarcomeric (SarMtCK) and ubiquitous (UbiMtCK). In some groups cytoplasmic dimers and mitochondrial octamers are found as well as a contiguous trimeric form, flagellar CK (flgCK). This latter isoform is found in the primitive-type spermatozoa of protochordates and certain protostome and deuterostome invertebrates. FlgCKs are myristolated and are found anchored to the flagellar membrane. AK isoforms are found as monomers, dimers, and contiguous dimers. AK is present in protozoans, protostome and deuterostome invertebrates, and protochordates, but not in craniates. Recently, a dimeric mitochondrial creatine kinase (MtCK) and a protoflagellar creatine kinase (protoflgCK) have been cloned and sequenced from the demosponge Tethya aurantia (Sona et al. 2004) and AK has been cloned and sequenced from the demosponge Suberites domuncula (Perovic-Ottstadt et al. 2005). Based on this earlier work we have surveyed all three classes of the poriferans, arguably the most primitive, basal metazoans, for the presence of CK and AK using spectrophotometric assays of tissue extracts as well as molecular biology protocols. Total RNA was isolated from three demosponges and three hexactinellids. RT-PCR was performed and cDNAs were amplified consisting of both mitochondrial and protoflagellar CK isoforms for two demosponges and two out of three hexactinellids tested. A gene for protoflgCK was amplified from a third hexactinellid. AKs were amplified from two demosponges and one hexactinellid. Our results show that AK is widely distributed in all three sponge classes. CK is present in two of the sponge classes and the genes for the mitochondrial and protoflagellar isoforms evolved prior to the divergence of the hexactinellids. It is highly probable that CK evolved in the sponges or in their immediate ancestors, the urmetazoans, or possibly even earlier. The deduced amino acid sequences from the amplified cDNAs were aligned and used in phylogenetic analyses along with CKs from other invertebrates and vertebrates and AKs from protozoans, invertebrates, and protochordates to elucidate the early evolution of the CK and AK gene families. Maximum Likelihood analysis using PHYLIP produced a CK tree with two distinct clades: mitochondrial and cytoplasmic-protoflagellar-flagellar. Using an outgroup composed of AKs, GKs, LKs, and TKs for evolutionary direction, our tree suggests that the ancestral CK dimer was MtCK-like and strongly suggests that the MtCK isoform was the first to evolve. Cyt, protoflg, and flgCK are closely related as well and phylogenetic evidence supports the idea that protoflgCKs are ancestral to flgCKs. The phylogenetic analysis completed in this effort provides the strongest evidence yet for a gene duplication event producing the true CytCKs and protoflgCKs. Maximum Likelihood analysis produced an AK tree with a base of protozoan, poriferan, and cnidarian AKs, and a clade of higher ecdysozoan and lophotrochozoan invertebrate AKs. To supplement this phylogenetic data and to further understand the early evolution of the CK isoforms, genomic DNA was isolated from Suberites ficus and the mitochondrial and protoflagellar genes were amplified by PCR. The gene organization data provides additional support that protoflgCKs are ancestral to flgCKs and that MtCKs are highly conserved in terms of their intron:exon organization. The present effort focuses on the distribution and early evolution of AK and CK in the poriferans, the pattern of gene duplication and divergence responsible for CK isoform diversity, and the development of intracellular targeting. The location and physiological roles of AK and CK will also be hypothesized and well as examining the utility of CK and AK as characters for sponge systematics. We will also explore structure-function relationships of these proteins and propose an evolutionary pathway for phosphagen kinases.