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The precise organization of pre- and postsynaptic terminals is crucial for normal synaptic function in the brain. In addition to its canonical role as a neurotrophin-3 receptor tyrosine kinase, postsynaptic TrkC promotes excitatory synapse organization through interaction with presynaptic receptor-type tyrosine phosphatase PTPσ. To isolate the synaptic organizer function of TrkC from its role as a neurotrophin-3 receptor, we generated mice carrying TrkC point mutations that selectively abolish PTPσ binding. The excitatory synapses in mutant mice had abnormal synaptic vesicle clustering and postsynaptic density elongation, more silent synapses, and fewer active synapses, which additionally exhibited enhanced basal transmission with impaired release probability. Alongside these phenotypes, we observed aberrant synaptic protein phosphorylation, but no differences in the neurotrophin signaling pathway. Consistent with reports linking these aberrantly phosphorylated proteins to neuropsychiatric disorders, mutant TrkC knock-in mice displayed impaired social responses and increased avoidance behavior. Thus, through its regulation of synaptic protein phosphorylation, the TrkC-PTPσ complex is crucial for the maturation, but not formation, of excitatory synapses in vivo., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

C-Mannosyl tryptophan (CMW), a unique glycosylated amino acid, is considered to be produced by degradation of C-mannosylated proteins in living organism. Although protein C-mannosylation is involved in the folding and secretion of substrate proteins, the pathophysiological function in the hematological system is still unclear. This study aimed to assess CMW in the human hematological disorders. The serum CMW levels of 94 healthy Japanese workers were quantified using hydrophilic interaction liquid chromatography. Platelet count was positively correlated with serum CMW levels. The clinical significance of CMW in thrombocytosis of myeloproliferative neoplasms (T-MPN) including essential thrombocythemia (ET) were investigated. The serum CMW levels of the 34 patients with T-MPN who presented with thrombocytosis were significantly higher than those of the 52 patients with control who had other hematological disorders. In patients with T-MPN, serum CMW levels were inversely correlated with anemia, which was related to myelofibrosis (MF). Bone marrow biopsy samples were obtained from 18 patients with ET, and serum CMW levels were simultaneously measured. Twelve patients with bone marrow fibrosis had significantly higher CMW levels than 6 patients without bone marrow fibrosis. Collectively, these results suggested that CMW could be a novel biomarker to predict MF progression in T-MPN., (© 2024. The Author(s).)

We previously reported the identification of a novel antimitotic agent with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-iodobenzohydrazide (code number NP-10). However, the mechanism(s) underlying the cancer cell-selective inhibition of mitotic progression by NP-10 remains unclear. Here, we identified NP-10-interacting proteins by affinity purification from HeLa cell lysates using NP-10-immobilized beads followed by mass spectrometry. The results showed that several mitosis-associated factors specifically bind to active NP-10, but not to an inactive NP-10 derivative. Among them, NUP155 and importin β may be involved in NP-10-mediated mitotic arrest. Because NP-10 did not show antitumor activity in vivo in a previous study, we synthesized 19 NP-10 derivatives to identify more effective NP-10-related compounds. HMI83-2, an NP-10-related compound with a Cl moiety, inhibited HCT116 cell tumor formation in nude mice without significant loss of body weight, suggesting that HMI83-2 is a promising lead compound for the development of novel antimitotic agents.

PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.

The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Xenopus mitogen-activated protein kinase kinase (MAPKK) previously inactivated with protein phosphatase 2A can be reactivated by serine phosphorylation catalyzed by a partially purified MAPKK kinase (MAPKK-K), and is phosphorylated by MAPK on a threonine residue. The sequence analysis of a threonine-phosphorylated tryptic peptide of Xenopus MAPKK from mature oocytes suggested that Thr388 is phosphorylated in vivo. A mutant MAPKK that has Thr388 changed to Ala (T388A-MAPKK) was not phosphorylated by purified MAPK, indicating that Thr388 is phosphorylated by MAPK. We then produced and analysed MAPKKs mutated at potential serine phosphorylation sites (S218A-MAPKK and S222A-MAPKK). The wild-type MAPKK (WT-MAPKKK), T388A-MAPKK and a kinase-deficient (K97S)-MAPKK were phosphorylated efficiently by MAPKK-Ks purified from Xenopus eggs, and WT-MAPKK and T388A-MAPKK became activated. In contrast, neither S218A-MAPKK nor S222A-MAPKK was phosphorylated and activated efficiently by the Xenopus MAPKK-Ks. Similarly, WT-MAPKK, but not S218A-MAPKK or S222A-MAPKK, was activated efficiently by an active Raf-1 immunoprecipitate. However, when the recombinant STE11, a putative MAPKK-K in S. cerevisiae, was used as a source of MAPKK-K, S218A-MAPKK as well as WT-MAPKK, but not S222A-MAPKK, was phosphorylated and activated. Furthermore, replacement of Ser222 with an acidic residue (S222E) elevated substantially the basal kinase activity of MAPKK, while replacement of Ser218 (S218E) did not. These results may suggest an essential role for Ser222 phosphorylation in activating Xenopus MAPKK.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.