scholarly journals A single tyrosine phosphorylation site in cortactin is important for filopodia formation in neuronal growth cones

2019 ◽  
Vol 30 (15) ◽  
pp. 1817-1833 ◽  
Author(s):  
Yuan Ren ◽  
Yingpei He ◽  
Sherlene Brown ◽  
Erica Zbornik ◽  
Michael J. Mlodzianoski ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Leading Edge ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Src Tyrosine Kinase ◽  
Actin Organization ◽  
Neuronal Growth Cones ◽  
Tyrosine Phosphorylation Site

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin–independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

Lipoprotein uptake by neuronal growth cones in vitro

Science ◽  
1987 ◽  
Vol 236 (4804) ◽  
pp. 959-962 ◽  
Author(s):  
M. Ignatius ◽  
E. Shooter ◽  
R. Pitas ◽  
R. Mahley
Keyword(s):  
Growth Cones ◽  
Neuronal Growth ◽  
Lipoprotein Uptake ◽  
Neuronal Growth Cones

scholarly journals Cofilin Regulates Filopodial Structure and Flexibility in Neuronal Growth Cones

2021 ◽  
Author(s):  
Ryan K. Hylton ◽  
Jessica Heebner ◽  
Michael Grillo ◽  
Matthew T Swulius
Keyword(s):  
Electron Tomography ◽  
Leading Edge ◽  
Growth Cones ◽  
Axonal Guidance ◽  
Neuronal Growth ◽  
Cellular Targets ◽  
Actin Networks ◽  
Motile Cells ◽  
Cryo Electron Tomography ◽  
Neuronal Growth Cones

Filopodia are actin-rich cytoskeletal protrusions at the leading edge of motile cells. In neuronal growth cones they function as antennae, guiding axonal growth toward the appropriate cellular targets. Proper brain development relies on robust axonal guidance mechanisms, so it is imperative to understand how the actin cytoskeleton functions in remodeling to meet the demands of growth cone exploration. Here we show by cryo-electron tomography and fluorescence imaging that filopodia in neuronal growth cones switch between fascin-linked and cofilin-decorated states, and that this transition regulates the exclusion of fascin from the cofilactin bundle at the filopodial base by hyper-twisting individual filaments and rearranging their packing. Additionally, we show that cofilactin bundles contribute to the flexibility of filopodial actin networks, thus, likely regulating the efficiency of targeted neurite outgrowth.

Targeting the Insulin-Like Growth Factor-I Receptor (IGF-IR) in Multiple Myeloma Cells Using Selective IGF-IR Tyrosine Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 639-639 ◽  
Author(s):  
Thomas Stromberg ◽  
Simon Ekman ◽  
Leonard Girnita ◽  
Johan Lennartsson ◽  
Ulf Hellman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Rpmi 8226 ◽  
Β Chain ◽  
Tyrosine Phosphorylation Site ◽  
Rtk Inhibitors

Abstract In multiple myeloma (MM) emerging evidence suggest the IGF-1R as an important mediator of tumor cell survival and thus resistance to cytotoxic therapy. Since IGF-1R is not an absolute requirement for maintenance of normal cell homeostasis, interfering with IGF-1R signaling at the receptor tyrosine kinase (RTK) level represents an attractive strategy to improve anti-cancer treatment. However, most IGF-1 RTK inhibitors do not fully discriminate between the IGF-1 RTK and the insulin RTK, i.e. are diabetogenic. Recently, members of the cyclolignan family have been shown to selectively inhibit the phosphorylation of the IGF-1R β-chain without down regulating the RTK activity of the insulin R1. The effect of two of these compounds, picropodophyllin (PPP) and deoxypodophyllotoxin (DPPT), was studied in vitro using a panel of nine MM cell lines and primary tumor cells from MM patients, and in vivo using the 5TMM mouse MM model2. Both IGF-1 RTK inhibitors effectively inhibited growth in all MM cell lines providing increased apoptosis and cell cycle arrest in the G2/M-phase. Notably, the two drug-resistant subclones of the MM cell line RPMI 8226/Dox40 (doxorubicin) and RPMI 8226/LR5 (melphalan), were highly sensitive to the IGF-1 RTK inhibitors. In addition, PPP and DPPT showed inhibitory effects on primary MM cells cultured in vitro with bone marrow stromal cells. Inhibition of the IGF-1 RTK with PPP has been shown to be non-competitive with ATP suggesting interference with the IGF-1R at the substrate level1. To identify tyrosine phosphorylation site(s) on the IGF-1R β-chain potentially regulated by the IGF-1 RTK inhibitors, IGF-1R extracted from RPMI 8226 cells was analyzed by tryptic phosphopeptide mapping following 32P-labeling and treatment with PPP or DPPT. Cleaved phosphopeptides were separated by thin layer chromatography and analyzed by a modified radio-Edman degradation. The results show that the IGF-1 RTK inhibitors do not down regulate any specific tyrosine phosphorylation site of the IGF-1R in MM cells. In vitro kinase assay suggests that PPP/DPPT-induced inhibition of the IGF-1R instead is conducted via a general down regulation of the tyrosine kinase activity of the receptor. Extraction of tyrosine phosphorylated proteins followed by electrophoresis and mass spectrometric analysis revealed additional signaling molecules affected by treatment with the IGF-1 RTK inhibitors e.g. impaired tyrosine phosphorylation of CDK1/cdc2, a cell cycle associated protein known as a potent regulator of apoptosis. Taken together, we show that treatment of MM cells with selective IGF-1 RTK inhibitors decreases survival/proliferation and affects the function of crucial intracellular signaling proteins, thus emphasizing the pivotal role for IGF-1R signaling in MM.

scholarly journals Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2745-2754 ◽  
Author(s):  
Alistair J. Standish ◽  
Jonathan J. Whittall ◽  
Renato Morona
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Capsular Polysaccharide ◽  
Regulatory Mechanism ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Regulatory Proteins ◽  
Amidase Activity ◽  
The Past

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

Localization of ganglioside 9-O-acetyl GD3 in point contacts of neuronal growth cones

2003 ◽  
Vol 57 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Erika M. A. Negreiros ◽  
Ana C. M. Leão ◽  
Marcelo F. Santiago ◽  
Rosalia Mendez-Otero
Keyword(s):  
Growth Cones ◽  
Point Contacts ◽  
Neuronal Growth ◽  
Neuronal Growth Cones
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