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P38 kinase regulates epidermal growth factor receptor downregulation and cellular migration.

ABSTRACT: Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.

SUBMITTER: Frey MR

PROVIDER: S-EPMC1698902 | biostudies-other | 2006 Dec

REPOSITORIES: biostudies-other

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p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration.

Frey Mark R MR Dise Rebecca S RS Edelblum Karen L KL Polk D Brent DB

The EMBO journal 20061130 24

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EG ...[more]

PMID: 17139251

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Project description:BACKGROUND:Spleen tyrosine kinase (SYK) is frequently upregulated in recurrent ovarian carcinomas, for which effective therapy is urgently needed. SYK phosphorylates several substrates, but their translational implications remain unclear. Here, we show that SYK interacts with EGFR and ERBB2, and directly enhances their phosphorylation. METHODS:We used immunohistochemistry and immunoblotting to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq and phosphoproteomic mass spectrometry to investigate the SYK-regulated EGF-induced transcriptome and downstream substrates. FINDINGS:Induced expression of constitutively active SYK130E reduced cellular response to EGFR/ERBB2 inhibitor, lapatinib. Expression of EGFRWT, but not SYK non-phosphorylatable EGFR3F mutant, resulted in paclitaxel resistance, a phenotype characteristic to SYK active ovarian cancers. In tumor xenografts, SYK inhibitor reduces phosphorylation of EGFR substrates. Compared to SYKWT cells, SYKKO cells have an attenuated EGFR/ERBB2-transcriptional activity and responsiveness to EGF-induced transcription. In ovarian cancer tissues, pSYK (Y525/526) levels showed a positive correlation with pEGFR (Y1187). Intense immunoreactivity of pSYK (Y525/526) correlated with poor overall survival in ovarian cancer patients. INTERPRETATION:These findings indicate that SYK activity positively modulates the EGFR pathway, providing a biological foundation for co-targeting SYK and EGFR. FUND: Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, NIH/NCI, Ovarian Cancer Research Foundation Alliance, HERA Women's Cancer Foundation and Roseman Foundation. Funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and eventually in the decision to submit the manuscript.

Project description:BackgroundEpidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.Methodology/principal findingsHere we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.Conclusions/significanceCortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.

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P38 kinase regulates epidermal growth factor receptor downregulation and cellular migration.

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p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration.

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