Dataset Information

Quantitative phosphoproteomic analysis of human Arl4A upon fibronectin stimulation

ABSTRACT: The extracellular matrix (ECM) fibronectin (FN) not only provides the framework for cell invasion and deposition of collagens and other ECMs from wound studies, but also guide collective tumor migration during cancer progression. p21-activated kinase 1 (Pak1) and Arf-like (Arl) GTPase Arl4A have previously been found to recruit each other on the plasm membrane to promote cell migration, and their plasma membrane translocation are robustly potentiated by FN. Under FN stimulation, we found that Pak1 kinase activity is required for Arl4A protein stabilization. How FN-induced Pak1 activity enhances Arl4A protein stability is unknown. Given that the Pak1-Arl4A interaction is direct, we hypothesized that alternation of Pak1-dependent phosphorylation on Arl4A leads to its protein stabilization upon FN stimulation. We herein applied the SILAC method to identify the phosphorylation sites on Arl4A upon FN treatment through MS analysis and found that S141/143 site(s) increased phosphorylation under FN stimulation. We further validated that phosphorylation on S143 of Arl4A and corresponding site on S144 of Arl4D (closest Arl member of Arl4A) are indeed dependent on Pak1 kinase and contribute to their protein stability. By dissecting the phosphorylation property on S143/S144 of Arl4A/D, we revealed an Arl4A/D specific chaperon protein HYPK as the underlying mechanism to mask and protect the phospho-form of Arl4A/D from fast proteasome degradation. This study provides how FN signaling cascade promotes cell migration on a molecular basis by regulating Arl4A/D phosphorylation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Chia-Jung Yu

LAB HEAD: Chia-Jung Yu

PROVIDER: PXD032397 | Pride | 2022-07-29

REPOSITORIES: Pride

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Publications

Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane.

Lin Ming-Chieh MC Yu Chia-Jung CJ Lee Fang-Jen S FS

Proceedings of the National Academy of Sciences of the United States of America 20220720 30

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4 ...[more]

PMID: 35857868

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Project description:Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma. Leucine to arginine substitution at amino acid position 858 (L858R) accounts for around 50% of EGFR-tyrosine kinase inhibitor (TKI) sensitizing mutations. A second site mutation in the gatekeeper residue (T790M) accounts for around 60% of acquired resistance to EGFR-TKIs. We sought to identify the immediate direct and indirect targets of these mutant EGFRs in lung adenocarcinoma and their modulation by erlotinib, the first generation, and most widely used EGFR-directed TKI. We undertook stable isotope labeling of amino acids in cell culture (SILAC), phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring L858R or L858R/T790M mutations and their modulation by erlotinib inhibition. Phosphorylation at the majority of phosphosites identified exhibited no change upon either EGF stimulation or erlotinib inhibition. Only around 7% of phosphosites identified and quantified showed increased phosphorylation upon EGF stimulation in either cell line. However, while phosphorylation at 61% of these phosphosites decreased upon erlotinib inhibition in the TKI sensitive H3255 cells, only 24% of such sites exhibited decreased phosphorylation upon erlotinib inhibition in the TKI resistant H1975 cells. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not the resistant cells include EGFR, Insulin receptor, HGF, MAPK, mTOR, p70S6K and JAK/STAT signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutive phosphorylated in these lung adenocarcinoma cells, but phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinase families, AGC, CAMK and STE were significantly enriched and those of proline directed kinase families, CMGC and CK were significantly depleted among substrates that exhibit increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma.

Dataset Information

Quantitative phosphoproteomic analysis of human Arl4A upon fibronectin stimulation

Publications

Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane.

Similar Datasets

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