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The Journal of Biological Chemistry Feb 1989Two forms of protein kinase activity were isolated from crude extracts of Streptococcus pyogenes and partially purified by ion exchange chromatography and affinity...
Two forms of protein kinase activity were isolated from crude extracts of Streptococcus pyogenes and partially purified by ion exchange chromatography and affinity chromatography. The phosphorylation activities were shown to be insensitive to cAMP, required the presence of divalent cations, and eluted from a Sephadex G-200 column with approximate molecular masses of 60 and 45 kDa, respectively. Both enzymes were capable of phosphorylating eukaryotic proteins and synthetic polypeptides in addition to endogenous and heterologous prokaryotic proteins at serine and tyrosine residues. Firm evidence for tyrosine kinase activity was obtained by the use of a tyrosine kinase-specific substrate, a 4:1 glutamate:tyrosine copolymer. Both protein kinases phosphorylated HPr, a phosphocarrier protein of the phosphotransferase system isolated from S. pyogenes and Bacillus stearothermophilus, but failed to phosphorylate HPr isolated from Escherichia coli. Both also phosphorylated a native polypeptide fragment (pep M24) as well as synthetic peptide copies of M protein, the major virulence determinant of group A streptococci. These results indicate that prokaryotic protein kinases are capable of phosphorylating eukaryotic proteins and suggest that the protein kinases of streptococci may play an important role not only in the phosphotransferase system but also in the virulence properties of these organisms.
Topics: Amino Acid Sequence; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Proteins; Carrier Proteins; Cations, Divalent; Kinetics; Molecular Weight; Peptides; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Streptococcus pyogenes
PubMed: 2521633
DOI: No ID Found -
PloS One 2014The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB...
The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.
Topics: Cell Differentiation; Chromatin; DNA-Binding Proteins; E2F1 Transcription Factor; Humans; K562 Cells; Nuclear Proteins; Phosphorylation; Retinoblastoma Protein; Serine; Threonine; U937 Cells
PubMed: 24466208
DOI: 10.1371/journal.pone.0086709 -
ELife Jul 2021Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the...
Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.
Topics: Cell Cycle Proteins; Cell Polarity; Cell Shape; Cytoskeleton; Phosphorylation; Protein Serine-Threonine Kinases; Ribonucleoproteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Signal Transduction; p21-Activated Kinases
PubMed: 34282727
DOI: 10.7554/eLife.67648 -
Proceedings of the National Academy of... Feb 2024To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) and CURTAIN-PTM...
To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) and CURTAIN-PTM (https://curtainptm.proteo.info) with an accompanying series of video tutorials (https://www.youtube.com/@CURTAIN-me6hl). These are designed to enable non-MS experts to interactively peruse volcano plots and deconvolute primary experimental data so that replicates can be visualized in bar charts or violin plots and exported in publication-ready format. They also allow assessment of overall experimental quality by correlation matrix and profile plot analysis. After making a selection of protein "hits", the user can analyze known domain structure, AlphaFold predicted structure, reported interactors, relative expression as well as disease links. CURTAIN-PTM permits analysis of all identified PTM sites on protein(s) of interest with selected databases. CURTAIN-PTM also links with the Kinase Library to predict upstream kinases that may phosphorylate sites of interest. We provide examples of the utility of CURTAIN and CURTAIN-PTM in analyzing how targeted degradation of the PPM1H Rab phosphatase that counteracts the Parkinson's LRRK2 kinase impacts cellular protein levels and phosphorylation sites. We also reanalyzed a ubiquitylation dataset, characterizing the PINK1-Parkin pathway activation in primary neurons, revealing data of interest not highlighted previously. CURTAIN and CURTAIN-PTM are free to use and open source, enabling researchers to share and maximize the impact of their proteomics data. We advocate that MS data published in volcano plot format be reported containing a shareable CURTAIN weblink, thereby allowing readers to better analyze and exploit the data.
Topics: Internet; Mass Spectrometry; Phosphorylation; Protein Processing, Post-Translational; Proteins; Proteomics; Software
PubMed: 38324566
DOI: 10.1073/pnas.2312676121 -
The New Phytologist Jun 2021Elongated hypocotyl5 (HY5) is a key transcription factor that promotes photomorphogenesis. Constitutive photomorphogenic1 (COP1)-Suppressor of phytochrome A-105 (SPA) E3...
Elongated hypocotyl5 (HY5) is a key transcription factor that promotes photomorphogenesis. Constitutive photomorphogenic1 (COP1)-Suppressor of phytochrome A-105 (SPA) E3 ubiquitin ligase complex promotes ubiquitination and degradation of HY5 to repress photomorphogenesis in darkness. HY5 is also regulated by phosphorylation at serine 36 residue. However, the kinase responsible for phosphorylation of HY5 remains unknown. Here, using extensive in vitro and in vivo biochemical, genetic, and photobiological techniques, we have identified a new kinase that phosphorylates HY5 and demonstrated the significance of phosphorylation of HY5 in Arabidopsis thaliana. We show that SPA proteins are the missing kinases necessary for HY5 phosphorylation. SPAs can directly phosphorylate HY5 in vitro, and the phosphorylated HY5 is absent in the spaQ background in vivo. We also demonstrate that the unphosphorylated HY5 interacts strongly with both COP1 and SPA1 and is the preferred substrate for degradation, whereas the phosphorylated HY5 is more stable in the dark. In addition, the unphosphorylated HY5 actively binds to the target promoters and is the physiologically more active form. Consistently, the transgenic plants expressing the unphosphorylated form of HY5 display enhanced photomorphogenesis. Collectively, our study revealed the missing kinase responsible for direct phosphorylation of HY5 that fine-tunes its stability and activity to regulate photomorphogenesis.
Topics: Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Gene Expression Regulation, Plant; Light; Phosphorylation; Phytochrome A; Ubiquitin-Protein Ligases
PubMed: 33686674
DOI: 10.1111/nph.17332 -
Current Protocols Sep 2022Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that...
Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).
Topics: Adenosine Triphosphate; Catalysis; Immunoprecipitation; Phosphoproteins; Phosphorylation
PubMed: 36135312
DOI: 10.1002/cpz1.539 -
The Journal of Biological Chemistry Aug 2007The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 of these serines are processively...
The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.
Topics: Amino Acid Sequence; Cloning, Molecular; Conserved Sequence; Evolution, Molecular; Humans; Molecular Sequence Data; Nuclear Proteins; Peptides; Phosphorylation; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine-Arginine Splicing Factors
PubMed: 17517895
DOI: 10.1074/jbc.M611305200 -
The Journal of Biological Chemistry Feb 1997Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the...
Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.
Topics: Amino Acid Sequence; Antibodies; Binding Sites; Cyclic AMP-Dependent Protein Kinases; Molecular Sequence Data; Phosphorylation; Protein Kinase C; Receptors, Amino Acid
PubMed: 9030583
DOI: 10.1074/jbc.272.8.5157 -
Biochimica Et Biophysica Acta. Gene... Nov 2022The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of...
The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.
Topics: Chromatin; Phosphorylation; Histones; DNA Damage; DNA
PubMed: 36280132
DOI: 10.1016/j.bbagrm.2022.194887 -
Journal of Neurochemistry Dec 2015Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin...
Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.
Topics: Animals; Arrestin; Decapodiformes; Eye Proteins; Light; Molecular Sequence Data; Phosphorylation; Photoreceptor Cells, Invertebrate; Rhodopsin; Serine; Signal Transduction; Vision, Ocular
PubMed: 26375013
DOI: 10.1111/jnc.13366