scholarly journals Tyrosine phosphorylation of type Iγ phosphatidylinositol phosphate kinase by Src regulates an integrin–talin switch

2003 ◽  
Vol 163 (6) ◽  
pp. 1339-1349 ◽  
Author(s):  
Kun Ling ◽  
Renee L. Doughman ◽  
Vidhya V. Iyer ◽  
Ari J. Firestone ◽  
Shawn F. Bairstow ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesions ◽  
Fold Increase ◽  
Signaling Proteins ◽  
Integrin Receptors ◽  
Dynamic Regulation ◽  
Phosphatidylinositol Phosphate ◽  
Precise Role ◽  
Phosphatidylinositol Phosphate Kinase

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover.

Regulation of Type II Phosphatidylinositol Phosphate Kinase by Tyrosine Phosphorylation in Bovine Rod Outer Segments†

Biochemistry ◽  
2001 ◽  
Vol 40 (15) ◽  
pp. 4550-4559 ◽  
Author(s):  
Zhong Huang ◽  
Xiao Xia Guo ◽  
Sherry X. Chen ◽  
Kathleen M. Alvarez ◽  
Michael W. Bell ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Rod Outer Segments ◽  
Type Ii ◽  
Outer Segments ◽  
Phosphatidylinositol Phosphate ◽  
Phosphatidylinositol Phosphate Kinase

Role of tyrosine kinase pathways in ETB receptor activation of NHE3

1996 ◽  
Vol 271 (3) ◽  
pp. C763-C771 ◽  
Author(s):  
T. S. Chu ◽  
H. Tsuganezawa ◽  
Y. Peng ◽  
A. Cano ◽  
M. Yanagisawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Integral Membrane Protein ◽  
Receptor Activation ◽  
Kinase C ◽  
Focal Adhesion Proteins ◽  
Etb Receptor

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.

scholarly journals Structural Basis for Phosphatidylinositol Phosphate Kinase Type Iγ Binding to Talin at Focal Adhesions

2004 ◽  
Vol 280 (9) ◽  
pp. 8381-8386 ◽  
Author(s):  
Jose M. de Pereda ◽  
Kate L. Wegener ◽  
Eugenio Santelli ◽  
Neil Bate ◽  
Mark H. Ginsberg ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Structural Basis ◽  
Phosphatidylinositol Phosphate ◽  
Phosphatidylinositol Phosphate Kinase

scholarly journals Bax signaling regulates palmitate-mediated apoptosis in C2C12 myotubes

2008 ◽  
Vol 295 (6) ◽  
pp. E1307-E1314 ◽  
Author(s):  
Jonathan M. Peterson ◽  
Yan Wang ◽  
Randall W. Bryner ◽  
David L. Williamson ◽  
Stephen E. Alway
Keyword(s):  
Insulin Resistance ◽  
Fold Increase ◽  
C2c12 Myotubes ◽  
Serum Starvation ◽  
Signaling Proteins ◽  
Induced Apoptosis ◽  
Apoptotic Effects

Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C2C12 myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C2C12 myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C2C12 myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in AktSer473 phosphorylation. Furthermore, treatment of the C2C12 myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C2C12 myotubes and that this effect corresponds to reductions in AktSer473 phosphorylation.

scholarly journals Golgi Fragmentation Is Associated with Ceramide-induced Cellular Effects

2005 ◽  
Vol 16 (3) ◽  
pp. 1555-1567 ◽  
Author(s):  
Wei Hu ◽  
Ruijuan Xu ◽  
Guofeng Zhang ◽  
Junfei Jin ◽  
Zdzislaw M. Szulc ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Hela Cells ◽  
Focal Adhesions ◽  
Fold Increase ◽  
Underlying Mechanism ◽  
Spectrometric Analysis ◽  
Integrin Receptors ◽  
Cellular Effects ◽  
Golgi Fragmentation ◽  
Hydrolysis Of

Ceramide has been shown to cause anoikis, a subtype of apoptosis due to inadequate cell adhesion. However, the underlying mechanism is unclear. Herein, we report that D-e-C6-ceramide (D-e-Cer), via generating sphingosine, disrupts the Golgi complex (GC), which is associated with various cellular effects, including anoikis. Treatment of HeLa cells with D-e-Cer caused cell elongation, spreading inhibition, rounding, and detachment before apoptosis (anoikis). In D-e-Cer–treated cells, glycosylation of β1 integrin in the GC was inhibited, thus its associated integrin receptors failed to translocate to the cell surface. Ceramide treatment also inhibited the reorganization of both microtubule and F-actin cytoskeletons, focal adhesions, and filopodia. These cellular effects were preceded by fragmentation of the Golgi complex. In contrast, L-e-C6-ceramide (L-e-Cer), the enantiomer of D-e-Cer, failed to induce these cellular effects. Mass spectrometric analysis revealed that treatment HeLa cells with D-e-Cer but not L-e-Cer caused a >50-fold increase in the levels of sphingosine, a product of hydrolysis of ceramide. Treatment with D-e-sphingosine and its enantiomer, L-e-sphingosine, caused massive perinuclear vacuolization, Golgi fragmentation, and cell rounding. Together, these results suggest that sphingosine generated from hydrolysis of ceramide causes the GC disruption, leading to various cellular effects.

Type Iγ phosphatidylinositol phosphate kinase targets and regulates focal adhesions

Nature ◽  
2002 ◽  
Vol 420 (6911) ◽  
pp. 89-93 ◽  
Author(s):  
Kun Ling ◽  
Renee L. Doughman ◽  
Ari J. Firestone ◽  
Matthew W. Bunce ◽  
Richard A. Anderson
Keyword(s):  
Focal Adhesions ◽  
Phosphatidylinositol Phosphate ◽  
Phosphatidylinositol Phosphate Kinase

scholarly journals Src-dependent NM2A tyrosine-phosphorylation regulates actomyosin dynamics

2020 ◽  
Author(s):  
Cláudia Brito ◽  
Francisco S. Mesquita ◽  
Daniel S. Osório ◽  
Joana Pereira ◽  
Neil Billington ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Biology ◽  
Bacterial Infections ◽  
Focal Adhesions ◽  
The Status ◽  
Fine Control ◽  
Actomyosin Cytoskeleton

AbstractNon-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that along with actin assembles into actomyosin filaments inside cells. NM2A is fundamental in cellular processes requiring force generation such as cell adhesion, motility and cell division, and plays important functions in different stages of development and during the progression of viral and bacterial infections. We previously identified at the motor domain of the NM2A, a novel Src-dependent tyrosine phosphorylation on residue 158 (pTyr158), which is promoted by Listeria monocytogenes infection. Despite the central role of NM2A in several cell biology processes, the pTyr at this specific residue had never been reported. Here we showed that LLO, a toxin secreted by Listeria, is sufficient to trigger NM2A pTyr158 by activating Src, which coordinates actomyosin remodeling. We further addressed the role of NM2A pTyr158 on the organization and dynamics of the actomyosin cytoskeleton and found that by controlling the activation of the NM2A, the status of the pTyr158 alters cytoskeletal organization, dynamics of focal adhesions and cell motility, without affecting NM2A enzymatic activity in vitro. Ultimately, by using Caenorhabditis elegans as a model to assess the role of this pTyr158in vivo, we found that the status of the pTyr158 has implications in gonad function and is required for organism survival under stress conditions. We conclude that the fine control of the NM2A pTyr158 is required for cell cytoskeletal remodeling and dynamics, and we propose Src-dependent NM2A pTyr158 as a novel layer of regulation of the actomyosin cytoskeleton.

scholarly journals Diacylglycerol metabolism in mast cells: a potential role in membrane fusion and arachidonic acid release.

1979 ◽  
Vol 150 (4) ◽  
pp. 1039-1044 ◽  
Author(s):  
D A Kennerly ◽  
T J Sullivan ◽  
P Sylwester ◽  
C W Parker
Keyword(s):  
Mast Cell ◽  
Arachidonic Acid ◽  
Mast Cells ◽  
Membrane Fusion ◽  
Cell Function ◽  
Fold Increase ◽  
Peritoneal Mast Cells ◽  
Acid Release ◽  
Precise Role

Purified rat peritoneal mast cells stimulated with the polycationic histamine-releasing agent compound 48/80 demonstrated a two- to four-fold increase in cellular levels of 1,2-diacylglycerol (DAG) within 1 min as detected by radioactive labeling and direct quantitation experiments. When 2-[1-14C]arachidonoyl-DAG was incubated in the presence of mast-cell homogenates, a rapid conversion to free arachidonate, and to a lesser extent, to monoacylglycerol, triglyceride, and phospholipid was observed. The release of arachidonate was proportional to the amount of broken-cell preparation added and the time of incubation, was prevented by preheating mast-cell preparations, and did not occur when 1-[1-14C]arachidonoyl-phosphatidylcholine was used as substrate, suggesting that the degradation was mediated by an enzyme with Dag-lipase activity. Although much work remains to be done to clarify the precise role of DAG in mast cells, DAG metabolism may be involved in secretion by generating substances which may faciliate membrane fusion and also in arachidonic acid-derived mediator formation by liberating esterified arachidonic acid from mast-cell lipids. Taken together, these studies indicate that the formation of DAG may play a central role in mast-cell function.

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