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We previously revealed the origin of mammalian simple-type glycogen synthase kinase interaction protein (GSKIP), which served as a scavenger and a competitor in the Wnt signaling pathway during evolution. In this study, we investigated the conserved and nonconserved regions of the composite-type GSKIP by utilizing bioinformatics tools, site-directed mutagenesis, and yeast two-hybrid methods. The regions were denoted as the pre-GSK3β binding site, which is located at the front of GSK3β-binding sites. Our data demonstrated that clustered mitochondria protein 1 (CLU1), a type of composite-type GSKIP that exists in the mitochondria of all eukaryotic organisms, possesses the protein known as domain of unknown function 727 (DUF727), with a pre-GSK3β-binding site and a mutant GSK3β-binding flanking region. Another type of composite-type GSKIP, armadillo repeat containing 4 (ARMC4), which is known for cilium movement in vertebrates, contains an unintegrated DUF727 flanking region with a pre-GSK3β-binding site (115SPxF118) only. In addition, the sequence of the GSK3β-binding site in CLU1 revealed that Q126L and V130L were not conserved, differing from the ideal GSK3β-binding sequence of simple-type GSKIP. We further illustrated two exceptions, namely 70 kilodalton heat shock proteins (Hsp70/DnaK) and Mitofilin in nematodes, that presented an unexpected ideal GSK3β-binding region with a pre-GSK3β sequence; this composite-type GSKIP could only occur in vertebrate species. Furthermore, we revealed the importance of the pre-GSK3β-binding site (118F or 118Y) and various mutant GSK3β-binding sites of composite-type GSKIP. Collectively, our data suggest that the new composite-type GSKIP starts with a DUF727 domain followed by a pre-GSK3β-binding site, with the subsequent addition of the GSK3β-binding site, which plays vital roles for CLU1, Mitofilin, and ARMC4 in mitochondria and Wnt signaling pathways during evolution.

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634~736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H(2)O(2)-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H(2)O(2) insult.