Src-mediated tyrosine phosphorylation of PRC1 and kinastrin/SKAP on the mitotic spindle

AbstractSrc-family tyrosine kinases (SFKs) play important roles in a number of signal transduction events during mitosis, such as spindle formation. A relationship has been reported between SFKs and the mitotic spindle; however, the underlying mechanisms remain unclear. We herein demonstrated that SFKs accumulated in the centrosome region at the onset of mitosis. Centrosomal Fyn increased in the G2 phase in a microtubule polymerization-dependent manner. A mass spectrometry analysis using mitotic spindle preparations was performed to identify tyrosine-phosphorylated substrates. Protein regulator of cytokinesis 1 (PRC1) and kinastrin/small kinetochore-associated protein (kinastrin/SKAP) were identified as SFK substrates. SFKs mainly phosphorylated PRC1 at Tyr-464 and kinastrin at Tyr-87. Although wild-type PRC1 is associated with microtubules, phosphomimetic PRC1 impaired the ability to bind microtubules. Phosphomimetic kinastrin at Tyr-87 also impaired binding with microtubules. Collectively, these results suggest that tyrosine phosphorylation of PRC1 and kinastrin plays a role in their delocalization from microtubules during mitosis.

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YTHDF2 alleviates cardiac hypertrophy via regulating Myh7 mRNA decoy

Cell & Bioscience ◽

10.1186/s13578-021-00649-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hongfei Xu ◽

Zhen Wang ◽

Miao Chen ◽

Wenting Zhao ◽

Tingting Tao ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Mass Spectrometry Analysis ◽

Immunofluorescent Staining ◽

Dependent Manner ◽

Protective Effects ◽

Spectrometry Analysis ◽

Pathological Cardiac Hypertrophy ◽

Therapeutic Mechanisms ◽

M6a Rna Methylation ◽

Underlying Mechanisms

Abstract Background Pathological cardiac hypertrophy is a major contributor of heart failure (HF), which seriously threatens human’s health world widely. Deregulation of m6A RNA methylation, and m6A methyltransferases and de-methyltransferases have been demonstrated to act essential roles in cardiac hypertrophy and HF. Here, we studied the potential roles and its underlying mechanisms of m6A Reader YTHDF proteins in HF. In this study, we constructed HF mouse model by transverse aortic constriction surgery. Primary cardiomyocytes were isolated and stimulated with isoproterenol (ISO) or phenylephrine (PHE) to induce myocardial hypertrophy. Results Through single-cell RNA-seq analysis, immunofluorescent staining, HE staining, Western blotting, and real time-PCR detections, we found that YTHDF2 mRNA and protein level, but not YTHDF1 or YTHDF3, was significantly increased during HF development. YTHDF2 overexpression could efficiently alleviate cardiac hypertrophy. Furthermore, through immunoprecipitation accompanied with mass spectrometry analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that ISO stimulation did not evidently affect YTHDF2-interacting proteins. However, ISO or PHE stimulation significantly increased YTHDF2 protein interacting with Myh7 (beta-myosin heavy chain) mRNA, an important cardiac hypertrophy marker, in an m6A-dependent manner. Knockdown of Myh7 or deletion of the YTH domain of YTHDF2 reversed the protective effects of YTHDF2 on cardiac hypertrophy. Finally, we found that ISO or PHE stimulation promoted YTHDF2 protein expression through enhancing Ythdf2 mRNA stability in an m6A-dependent manner in cardiomyocytes. Conclusions Overall, our results indicate that the m6A Reader YTHDF2 suppresses cardiac hypertrophy via Myh7 mRNA decoy in an m6A-dependent manner. This study highlights the functional importance of YTHDF2-dependent cardiac m6A mRNA regulation during cardiac hypertrophy, and provides a novel mechanistic insight into the therapeutic mechanisms of YTHDF2.

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GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

Frontiers in Neuroscience ◽

10.3389/fnins.2021.692217 ◽

2021 ◽

Vol 15 ◽

Author(s):

Guokun Zhou ◽

Xiang-ming Zha

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Neurological Diseases ◽

Hippocampal Slices ◽

Signaling Cascades ◽

Dependent Manner ◽

Cyclin Dependent Kinase ◽

Ataxia Telangiectasia Mutated ◽

Agc Kinases ◽

Profiling Analysis

Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68−/− slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.

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Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules.

The Journal of Cell Biology ◽

10.1083/jcb.97.6.1918 ◽

1983 ◽

Vol 97 (6) ◽

pp. 1918-1924 ◽

Cited By ~ 122

Author(s):

C Keith ◽

M DiPaola ◽

F R Maxfield ◽

M L Shelanski

Keyword(s):

Mitotic Spindle ◽

Calcium Ion ◽

Molar Ratio ◽

Free Calcium ◽

Local Concentration ◽

Dependent Manner ◽

Calcium Dependent ◽

Molar Ratios ◽

Microtubule Polymerization ◽

Full Complement

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.

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Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR

Biochemical Journal ◽

10.1042/bcj20170520 ◽

2018 ◽

Vol 475 (5) ◽

pp. 981-1002 ◽

Cited By ~ 9

Author(s):

Jie Li ◽

Hiroki Shima ◽

Hironari Nishizawa ◽

Masatoshi Ikeda ◽

Andrey Brydun ◽

...

Keyword(s):

Mitotic Spindle ◽

Isotope Labeling ◽

Binding Activity ◽

Mass Spectrometry Analysis ◽

Spindle Orientation ◽

Mitotic Spindles ◽

Spectrometry Analysis ◽

Dna Binding Activity ◽

Ejection Force ◽

M Phase

The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.

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Quantitative proteomics identifies FOLR1 to drive sorafenib resistance via activating autophagy in hepatocellular carcinoma cells

Carcinogenesis ◽

10.1093/carcin/bgab019 ◽

2021 ◽

Author(s):

Hongwei Chu ◽

Changqing Wu ◽

Qun Zhao ◽

Rui Sun ◽

Kuo Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Folate Receptor ◽

Mass Spectrometry Analysis ◽

Label Free ◽

Spectrometry Analysis ◽

Underlying Mechanisms ◽

Related Proteins ◽

Hcc Cells

Abstract Sorafenib is commonly used to treat advanced human hepatocellular carcinoma (HCC). However, clinical efficacy has been limited by drug resistance. In this study, we used label-free quantitative proteomic analysis to systematically investigate the underlying mechanisms of sorafenib resistance in HCC cells. A total of 1709 proteins were confidently quantified. Among them, 89 were differentially expressed, and highly enriched in the processes of cell-cell adhesion, negative regulation of apoptosis, response to drug and metabolic processes involving in sorafenib resistance. Notably, folate receptor α (FOLR1) was found to be significantly upregulated in resistant HCC cells. In addition, in-vitro studies showed that overexpression of FOLR1 decreased the sensitivity of HCC cells to sorafenib, whereas siRNA-directed knockdown of FOLR1 increased the sensitivity of HCC cells to sorafenib. Immunoprecipitation-mass spectrometry analysis suggested a strong link between FOLR1 and autophagy related proteins. Further biological experiments found that FOLR1-related sorafenib resistance was accompanied by the activation of autophagy, whereas inhibition of autophagy significantly reduced FOLR1-induced cell resistance. These results suggest the driving role of FOLR1 in HCC resistance to sorafenib, which may be exerted through FOLR1-induced autophagy. Therefore, this study may provide new insights into understanding the mechanism of sorafenib resistance.

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Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽

10.1182/blood.v104.11.3541.3541 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3541-3541

Author(s):

Swaminathan Murugappan ◽

Haripriya Shankar ◽

Satya Kunapuli

Keyword(s):

Tyrosine Kinase ◽

Intracellular Calcium ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Human Platelets ◽

Dependent Manner ◽

Protein Signaling ◽

Glycoprotein Vi ◽

Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

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Arginine Methylation Of RBM15 By PRMT1 Controls Alternative Splicing Of Genes Involved In Megakaryocyte Differentiation

Blood ◽

10.1182/blood.v122.21.2443.2443 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2443-2443

Author(s):

Xinyang Zhao ◽

Li Zhang ◽

Rui Wang ◽

Ngoc Tung Trans ◽

Hairui Su ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Chromosome Translocation ◽

Arginine Methylation ◽

Mass Spectrometry Analysis ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Spectrometry Analysis ◽

Megakaryocyte Differentiation

Abstract More than 90% of under one year old infants with acute megakaryoblastic leukemia (AMKL) have chromosome translocation t(1;22)(p13;q13) with RBM15 fused to MKL1. RBM15 encodes an RNA binding protein important for hematopoietic stem cell self-renewal and differentiation. In agreement with its roles in AMKL, RBM15 is required for normal megakaryocyte differentiation. We found that higher expression of PRMT1 (Protein Arginine Methyltransferase) is commonly seen in M7 leukemia patient samples than other types of myeloid leukemia and that RBM15 is a bona fide methylation target for PRMT1. Using mass spectrometry analysis, we mapped the PRMT1 mediated mono-methylated site. The enzymatic activity of the PRMT1 V2 isoform is required for RBM15 degradation, as both shRNA molecules knocking down PRMT1 and small chemical PRMT1 inhibitors stabilize the RBM15 protein. Mutation of the methylation site to lysine blocks the ubiquitylation mediated degradation. Thus the degradation is a methylation dependent process. We identified the E3 ligase responsible for the degradation. Down-regulation of the RBM15 protein changes the isoform ratio of genes including GATA1 critical megakaryocyte differentiation. We found that RBM15 regulates its interaction with SF3B1A in methylation dependent manner during alternative splicing of GATA1 pre-mRNA. Thus, via methylation triggered RBM15 degradation, the megakaryocyte progenitor cells maintain a delicate balance between differentiation and proliferation by keeping the proper ratio of GATA1s and GATA1-full length mRNA. SF3B1A has been shown to be mutated in myeloid dysplasia syndrome and in several different types of leukemia. Methylation by PRMT1 links the two types of leukemic genes into a single pathway. Our results imply that targeting PRMT1/RBM15 pathway might be a potential therapy for AMKL and other blood malignancies. Disclosures: No relevant conflicts of interest to declare.

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DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612980114 ◽

2017 ◽

Vol 114 (28) ◽

pp. 7408-7413 ◽

Cited By ~ 7

Author(s):

Yan Jin ◽

Yaohui Chen ◽

Shimin Zhao ◽

Kun-Liang Guan ◽

Yuan Zhuang ◽

...

Keyword(s):

Complex Formation ◽

Germ Line ◽

Mechanistic Explanation ◽

Mass Spectrometry Analysis ◽

Physical Interaction ◽

Spectrometry Analysis ◽

Culture Cells ◽

Underlying Mechanisms

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA–PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA–PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA–PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA–PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA–PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA–PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA–PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

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Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase- dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes

Blood ◽

10.1182/blood.v82.4.1212.bloodjournal8241212 ◽

1993 ◽

Vol 82 (4) ◽

pp. 1212-1220 ◽

Cited By ~ 2

Author(s):

GL Schieven ◽

JM Kirihara ◽

DE Myers ◽

JA Ledbetter ◽

FM Uckun

Keyword(s):

Ionizing Radiation ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Reactive Oxygen ◽

Lymphoid Cells ◽

Dependent Manner ◽

Reactive Oxygen Intermediates ◽

Nf Kappa B ◽

Oxygen Intermediates

We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.

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Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.186 ◽

1995 ◽

Vol 15 (1) ◽

pp. 186-197 ◽

Cited By ~ 135

Author(s):

S Richard ◽

D Yu ◽

K J Blumer ◽

D Hausladen ◽

M W Olszowy ◽

...

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Src Family Kinases ◽

Src Family Kinase ◽

Sh3 Domains ◽

Sh2 Domains ◽

Src Family Tyrosine Kinases

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

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