Transient exposure of a buried phosphorylation site in an autoinhibited protein

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

Nature Communications ◽

10.1038/s41467-021-23116-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Javier Emperador-Melero ◽

Man Yan Wong ◽

Shan Shan H. Wang ◽

Giovanni de Nola ◽

Hajnalka Nyitrai ◽

...

Keyword(s):

Phase Separation ◽

Active Zone ◽

Neurotransmitter Release ◽

Hippocampal Neurons ◽

Phosphorylation Site ◽

Double Knockout ◽

Zone Structure ◽

Presynaptic Nerve Terminal ◽

Condensate Formation ◽

And Function

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

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Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)89875-1 ◽

1984 ◽

Vol 259 (22) ◽

pp. 14184-14189

Author(s):

J P Albanesi ◽

H Fujisaki ◽

E D Korn

Keyword(s):

Active Site ◽

Phosphorylation Site

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Cardiac-specific phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase is conserved in the brain ryanodine receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42924-9 ◽

1992 ◽

Vol 267 (7) ◽

pp. 4963-4967

Author(s):

D.R. Witcher ◽

B.A. Strifler ◽

L.R. Jones

Keyword(s):

Protein Kinase ◽

Ryanodine Receptor ◽

Phosphorylation Site ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

The Brain

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EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

Cell Death and Disease ◽

10.1038/s41419-021-03721-9 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Yiming He ◽

Mingxi Gan ◽

Yanan Wang ◽

Tong Huang ◽

Jianbin Wang ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Cell Cycle Progression ◽

Potential Role ◽

Target Genes ◽

Nuclear Translocation ◽

Phosphorylation Site ◽

Promoter Regions ◽

Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

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A deep learning based approach for prediction of Chlamydomonas reinhardtii phosphorylation sites

Scientific Reports ◽

10.1038/s41598-021-91840-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Niraj Thapa ◽

Meenal Chaudhari ◽

Anthony A. Iannetta ◽

Clarence White ◽

Kaushik Roy ◽

...

Keyword(s):

Deep Learning ◽

Chlamydomonas Reinhardtii ◽

Protein Phosphorylation ◽

Short Term Memory ◽

Phosphorylation Site ◽

Specific Protein ◽

Training Dataset ◽

Phosphorylation Sites ◽

Site Prediction ◽

Model Combining

AbstractProtein phosphorylation, which is one of the most important post-translational modifications (PTMs), is involved in regulating myriad cellular processes. Herein, we present a novel deep learning based approach for organism-specific protein phosphorylation site prediction in Chlamydomonas reinhardtii, a model algal phototroph. An ensemble model combining convolutional neural networks and long short-term memory (LSTM) achieves the best performance in predicting phosphorylation sites in C. reinhardtii. Deemed Chlamy-EnPhosSite, the measured best AUC and MCC are 0.90 and 0.64 respectively for a combined dataset of serine (S) and threonine (T) in independent testing higher than those measures for other predictors. When applied to the entire C. reinhardtii proteome (totaling 1,809,304 S and T sites), Chlamy-EnPhosSite yielded 499,411 phosphorylated sites with a cut-off value of 0.5 and 237,949 phosphorylated sites with a cut-off value of 0.7. These predictions were compared to an experimental dataset of phosphosites identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS) in a blinded study and approximately 89.69% of 2,663 C. reinhardtii S and T phosphorylation sites were successfully predicted by Chlamy-EnPhosSite at a probability cut-off of 0.5 and 76.83% of sites were successfully identified at a more stringent 0.7 cut-off. Interestingly, Chlamy-EnPhosSite also successfully predicted experimentally confirmed phosphorylation sites in a protein sequence (e.g., RPS6 S245) which did not appear in the training dataset, highlighting prediction accuracy and the power of leveraging predictions to identify biologically relevant PTM sites. These results demonstrate that our method represents a robust and complementary technique for high-throughput phosphorylation site prediction in C. reinhardtii. It has potential to serve as a useful tool to the community. Chlamy-EnPhosSite will contribute to the understanding of how protein phosphorylation influences various biological processes in this important model microalga.

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Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53319-6 ◽

1993 ◽

Vol 268 (9) ◽

pp. 6791-6799

Author(s):

P.W. Jurutka ◽

J.C. Hsieh ◽

P.N. MacDonald ◽

C.M. Terpening ◽

C.A. Haussler ◽

...

Keyword(s):

Vitamin D ◽

Amino Acid ◽

Vitamin D Receptor ◽

Phosphorylation Site ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Intact Cells ◽

Predominant Amino Acid

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Molecular Characterization and Phylogenetic Analysis of MADS-Box Gene VroAGL11 Associated with Stenospermocarpic Seedlessness in Muscadine Grapes

Genes ◽

10.3390/genes12020232 ◽

2021 ◽

Vol 12 (2) ◽

pp. 232

Author(s):

M Atikur Rahman ◽

Subramani P Balasubramani ◽

Sheikh M Basha

Keyword(s):

Phylogenetic Analysis ◽

Phosphorylation Site ◽

Orthologous Gene ◽

Mads Box ◽

Vitis Species ◽

Vitis Rotundifolia ◽

Seed Content ◽

Putative Phosphorylation Site ◽

Mads Box Gene ◽

Muscadine Grapes

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar “Jane Bell” containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology.

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