Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligaselike (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase. The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity. Tubulin, which is the major protein subunit of MT fibers, exhibits polymorphic protein variation and undergoes a variety of unique post-translational modifications at its C-terminal tail, such as detyrosination/tyrosination (1, 2), polyglycylation (3), and polyglutamylation (4). These modifications regulate tubulin and MT function. Polyglutamylation enzymes add multiple glutamate moieties to specific glutamate residues within substrate proteins. Physiological roles of tubulin polyglutamylation for MT-related processes were reported recently (5, 6) and reviewed by Ikegami and Setou (7).Polyglutamylase enzymes have recently been identified as belonging to the tubulin tyrosine ligase-like (TTLL) protein family (8 -10) (supplemental Fig. 1). In contrast, the enzyme(s) underlying deglutamylation have not been discovered yet, although such proteins are suggested to be present in mouse brain neurons and in cultured cells (11,12).In this study, we employed the genetically tractable nematode, Caenorhabditis elegans, to identify genes that regulate tubulin polyglutamylation/deglutamylation. Specifically, we investigated tubulin post-translational modification in C. elegans sensory cilia, which are MT-based organelles that extend from the distal dendrite tips of 60 sensory neurons found in sensory organs named amphid (head) and phasmid (tail) sensilla (see Fig. 1A). In this system, we identified a cytosolic carboxypeptidase (ccpp) gene, ccpp-6, as a candidate tubulin deglutamylase gene, whose functional properties are opposite ...
Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/ threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.
Background: Hyperphosphorylated Tau is a component of neurofibrillary tangles, the pathological hallmark in brains with tauopathies. Results: Pin1 binds phospho-Tau and stimulates its dephosphorylation at Cdk5-mediated phosphorylation sites. Conclusion: Efficient Tau dephosphorylation at Alzheimer-related sites requires Pin1 activity, thereby preventing Tau hyperphosphorylation. Significance: Disruption of Pin1-dependent facilitation of Tau dephosphorylation may be a critical mechanism underlying the etiology of tauopathies.
Apoptosis‐associated tyrosine kinase 1 (AATYK1), also named LMTK1, was previously isolated as an apoptosis‐related gene from 32Dcl3 myeloid precursor cells, but its precise function remains unknown. AATYK1A, an isoform without a transmembrane domain, is highly expressed in neurons. We identified palmitoylation of AATYK1A at three N‐terminal cysteine residues in cortical cultured neurons and COS‐7 cells and found that palmitoylation determined localization of AATYK1A to the transferrin receptor‐positive recycling endosomes. Further, we identified the tyrosine kinase Src as a novel AATYK1A‐interacting protein. Src and Fyn phosphorylated AATYK1A at tyrosines 25 and 46 in a palmitoylation‐dependent manner. The association of AATYK1A with Src in endosomes was also found to be palmitoylation‐dependent. These results indicate that palmitoylation is a critical factor not only for the subcellular localization of AATYK1A but also for its interaction with Src.
Background: FilGAP is a Rac GTPase-activating protein, but regulation via phosphorylation has not been previously characterized. Results: Phosphorylation of FilGAP at serine 402 is necessary to activate FilGAP to suppress cell spreading on fibronectin. Conclusion: Serine 402 is a critical phosphorylation site to regulate FilGAP activity. Significance: This study showed that regulation of FilGAP by phosphorylation may play a role in integrin-mediated cell adhesion on fibronectin.
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