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:Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. Exogenous expression of human CD9 by CD9-adenoviral transduction led to increases in neointima. CD9 expression significantly augmented PI-3 kinase dependent Akt phosphorylation with concurrent adhesion to extracellular matrix protein fibronectin (FN). Furthermore,enhanced Akt phosphorylation was attenuated by anti-CD9 mAb7 binding. As multiple factors contribute to the regulation of VSMC phenotype, our aim was to establish whether increased CD9 expression would induce changes in the expression of other genes associated with VSMC proliferation and motility. RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data. Analysis of this data showed the regulation of some key genes involved in the modulation of VSMC motility and proliferation. Experiment Overall Design: RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data.

			Action	DRS
	Arl4A_FN_phospho-1.msf	Msf
	Arl4A_FN_phospho-1.raw	Raw
	Arl4A_FN_phospho-1_swap.msf	Msf
	Arl4A_FN_phospho-1_swap.raw	Raw
	Arl4A_FN_phospho-2.msf	Msf