scholarly journals Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth

2020 ◽  
Vol 113 (6) ◽  
pp. 1053-1069 ◽  
Author(s):  
Ramona Märker ◽  
Bernhard Blank‐Landeshammer ◽  
Anna Beier‐Rosberger ◽  
Albert Sickmann ◽  
Ulrich Kück
Keyword(s):  
Sexual Development ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Polarized Growth ◽  
Pak Kinase

scholarly journals iTRAQ-based proteomic and phosphoproteomic analyses of STRIPAK mutants from the fungus Sordaria macrospora identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important for sexual development and polarized growth

2019 ◽  
Author(s):  
R Märker ◽  
B Blank-Landeshammer ◽  
A Beier-Rosberger ◽  
A Sickmann ◽  
U Kück
Keyword(s):  
Sexual Development ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Phosphorylation Site ◽  
Absolute Quantification ◽  
Serine Phosphorylation ◽  
Polarized Growth ◽  
Sordaria Macrospora ◽  
Eukaryotic Microorganisms ◽  
The Impact

SummaryThe highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation of developmental proteins in eukaryotic microorganisms, animals, and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild-type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase (PAK) family. Functional characterization of the Δcla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phospho-mimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

scholarly journals Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sung Hoon Choi ◽  
Hee-Sub Yoon ◽  
Shin-Ae Yoo ◽  
Sung Ho Yun ◽  
Joo-Hee Park ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Phosphorylation Site ◽  
Aurora Kinase ◽  
Serine Phosphorylation ◽  
Flight Mass Spectrometry ◽  
Iκb Kinase ◽  
Orbitrap Mass Spectrometer ◽  
Nf Kappab

Abstract Background Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells. Methods Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive). Results We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells. Conclusions Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.

scholarly journals Increased oncogenic potential of ErbB is associated with the loss of a COOH-terminal domain serine phosphorylation site.

1992 ◽  
Vol 267 (12) ◽  
pp. 7967-7970
Author(s):  
S.J. Theroux ◽  
C Taglienti-Sian ◽  
N Nair ◽  
J.L. Countaway ◽  
H.L. Robinson ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Oncogenic Potential ◽  
Terminal Domain

scholarly journals The Actin-Binding Protein UNC-115/abLIM Controls Formation of Lamellipodia and Filopodia and Neuronal Morphogenesis in Caenorhabditis elegans

2005 ◽  
Vol 25 (12) ◽  
pp. 5158-5170 ◽  
Author(s):  
Yieyie Yang ◽  
Erik A. Lundquist
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Axon Pathfinding ◽  
Neuronal Morphogenesis ◽  
Actin Binding Protein ◽  
C Elegans

ABSTRACT The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.

Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Large Family ◽  
Adaptor Proteins ◽  
Serine Phosphorylation ◽  
Binding Motif ◽  
Double Knockout ◽  
Coated Pits ◽  
Knockout Mutants ◽  
Golgi Network

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

scholarly journals Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

2003 ◽  
Vol 278 (30) ◽  
pp. 28123-28129 ◽  
Author(s):  
Josef Köck ◽  
Michael Kann ◽  
Gerhard Pütz ◽  
Hubert E. Blum ◽  
Fritz von Weizsäcker
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Duck Hepatitis B Virus ◽  
Virus Core ◽  
Duck Hepatitis ◽  
B Virus

Identification of a Human Akt3 (Protein Kinase B γ) Which Contains the Regulatory Serine Phosphorylation Site

1999 ◽  
Vol 257 (3) ◽  
pp. 906-910 ◽  
Author(s):  
Kaname Nakatani ◽  
Hiroshi Sakaue ◽  
Devon A. Thompson ◽  
Ronald J. Weigel ◽  
Richard A. Roth
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Phosphorylation Site ◽  
Serine Phosphorylation

scholarly journals Molecular cloning of a novel N-terminal variant of annexin II from rat basophilic leukaemia cells

1994 ◽  
Vol 302 (2) ◽  
pp. 425-428 ◽  
Author(s):  
A L Upton ◽  
S E Moss
Keyword(s):  
Single Gene ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Annexin Ii ◽  
Cdna Clones ◽  
Potential Phosphorylation Site ◽  
Plasmid Library ◽  
Alternatively Spliced ◽  
High Level ◽  
Species Hybridization

Rat annexin II cDNA clones were isolated from a rat basophilic leukaemia cell plasmid library by cross-species hybridization with a mouse probe, and fully sequenced using the dideoxy-chain-termination method. Alignment of the derived amino-acid sequence with those of other mammalian annexin II species revealed a high level of conservation, characteristic of the annexin family of proteins. One of the cDNAs isolated contained an additional six nucleotides close to the N-terminus, lying in-frame and at a point corresponding to an intron/exon boundary in the human annexin II gene. As the two rat cDNAs were identical apart from the six nucleotide insert, it is likely that these represent alternatively spliced transcripts of a single gene, rather than the products of two separate genes. The six nucleotides encode serine-glutamine and therefore introduce an additional potential phosphorylation site into a region already containing one tyrosine and two serine phosphorylation sites. The discovery of this novel annexin II variant may have important implications both for p11 binding and for regulation of annexin II function by phosphorylation.

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