Creatine kinase (CK) is one of the eight members of the family of phosphagen kinases, enzymes that play an important role in energy metabolism of cells displaying high and variable rates of ATP turnover such as muscle fibers, photoreceptors, neurons, transport epithelia and spermatozoa. CK catalyses the reversible transfer of the phosphoryl group from creatine phosphate (CP) to ADP yielding ATP. In the vertebrates CK exists as two cytoplasmic isoforms namely muscle (M) and brain (B), which are capable of forming homo- (MM, BB) and heterodimers (MB). In striated skeletal muscle fibers, a small but significant fraction of MM-CK is localized near the M-line in the sarcomere and near sarcoplasmic reticulum (SR) Ca+2-ATPases while some BB-CK in neurons is localized on the plasma membrane near the Na+:K+-ATPases. In the vertebrates the so-called sarcomeric mitochondrial CK (sarMiCK) and ubiquitous mitochondrial CK (ubiMiCK) isoforms of CK are targeted to the mitochondrial intermembrane space where they exist in an octamer-dimer equilibrium. In the primitive-type spermatozoa of certain protochordates and protostome and deuterostome invertebrates, yet another type of CK is present which is localized in the flagellar membrane via a myristoylate anchor. This flagellar CK (flgCK) consists of three fused, complete CK domains and was the result of a gene duplication-fusion event followed by unequal crossing over.
The available evidence indicates that all CK isoforms are targeted to some extent to specific intracellular compartments and are often found to be closely associated with the sites of ATP generation and utilization. How early did this commitment to intracellular targeting take place? This thesis focuses on the cloning and expression of three CKs, denoted CK1, CK2 and CK3, from the sponge Tethya aurantia, a representative of the oldest, extant group of metazoan (multi-cellular) animals. Sequence analyses showed that CK2 most closely resembles mitochondrial CKs, and phylogenetic analyses using Neighbor Joining (NJ) revealed that CK2 forms the base of the mitochondrial clade whereas CK1 and CK3 form the base of a large cluster consisting of the flagellar CKs. CK1 and CK3 are very similar to each other. The cDNAs for CK1 and CK3 and three constructs of CK2 in which the sequence for the targeting peptide was deleted at different cleavage sites (termed CK2/1-3) were expressed in E. coli. All constructs yielded significant soluble CK activity that was partially purified and then subjected to size exclusion chromatography. CK1 and CK3 eluted as dimers. Given their position in the NJ tree and the fact that these CKs exist as dimers, these proteins were termed protoflagellar CKs.
Tethya CK2 is clearly a mitochondrial CK based on the presence of a mitochondrial targeting peptide and its position in the NJ tree. Surprisingly, the three mitochondrial constructs (CK2/1-3) were also found to be dimers in contrast to all known invertebrate and vertebrate mitochondrial CKs that are primarily octameric or mixtures of octamers and dimers. A conserved tryptophan residue (Trp264 in Chicken sarMiCK) has been implicated in a number of studies as being important for octamer formation. Tethya CK2 has a Tyr residue at this equivalent position. In order to evaluate the role of this highly conserved tryptophan residue in the quaternary structure of mitochondrial CKs, site- directed mutagenesis of the Tyr à Trp on the three CK2 constructs was performed and the oligomeric state determined using Dynamic Light Scattering (DLS). Earlier mutagenesis studies as well as recent unpublished work from our lab have shown that the mutation of this tryptophan with a conserved amino acid leads to the destabilization and in some cases complete disruption of the octamer. Interestingly the Tyr à Trp mutation on the three CK2 constructs did not lead to the formation of an octamer. It appears that although the absolutely conserved tryptophan residue is important for octamer stabilization, there are other forces involved that lead to the formation of a functional octamer. Thus, T. aurantia mitochondrial CK appears to be missing at least one, possibly many, of the structural elements necessary for octamerization and, as such, constitutes an early step in the evolution of MiCKs and potentially may be a useful experimental vehicle for elucidating the evolutionary steps leading to octamer formation in this key metabolic enzyme.