Figure 4. Phosphoproteomics analysis of cells invading HDFC matrix reveals complex downstream signaling. (A) Comparison of whole-genome microarray expression profiles for MDA-MB-231 cells with or without serum, or invading gelatin versus HDFC matrices, based on data pooled from five independent experiments for each condition. (B) Localization of activated β1 integrin to invadopodia of the MDA-MB-231 adherent to HDFC at the absence of serum. Invadopodia are yellow dots with colocalized actin and cortactin. (C) Localization of activated β1 integrin to FAs of MDA-MB-231 cells adherent to gelatin matrix in the absence of serum, showing poor invadopodia formation. (D) Comparative analysis of protein expression levels in MDA-MB-231 cells on HDFC versus cells on gelatin at the absence of serum. Cell lysates from each sample were labeled with specific iTRAQ labels to compare amounts of each protein (protein rank) identified by MS between HDFC and gelatin samples. (E) Numbers of unique and shared phosphoproteins and phosphopeptides in MDA-MB-231 carcinoma cells invading HDFC (green) compared with 2D gelatin (red) matrices in serum-free medium as identified by phosphoproteomics. (F) Quantification of types of phosphorylation sites identified by phosphoproteomics analysis. (G) Proposed signaling network associated with integrin-dependent induction of abundant invadopodia in carcinoma cells invading HDFC matrix. All identified phosphoproteins and their phosphosites are listed in Table S1. Open boxes indicate phosphoproteins identified in both HDFC and gelatin samples with the same phosphorylation sites by comparison of phosphopeptides. Gray boxes denote phosphoproteins identified in HDFC versus gelatin samples with different phosphorylation sites. Orange boxes indicate phosphoproteins unique to the HDFC matrix. Proteins without phosphorylation changes, such as Rac1, Cdc42, RhoA, and αβ integrin, are added to clarify the signaling context of proteins identified by phosphoproteomics. Although not regulated by phosphorylation, migfilin is depicted in the network because it is known to bind directly to both kindlin2 and filamin A. Migfilin and the other proteins highlighted by thick green outlines were verified experimentally in this study to play a role in invadopodia regulation by siRNA knockdown. Solid lines indicate known direct physical binding between proteins. Dashed lines indicate indirect interactions involving intermediate partners. Black arrows at the ends of lines indicate proteins known to stimulate the downstream signaling partner, lines with inhibition symbol indicate down-regulation of activity of the downstream signaling partner, lines with an inhibition symbol plus a black arrow indicate both potential activation and inhibition, and open arrows denote stimulation of the cellular process. Abbreviations used in this figure: ROCK, Rho-associated protein kinase; MLCP, myosin light-chain phosphatase; MLCK, myosin light-chain kinase; MPRIP, myosin phosphatase Rho-interacting protein; ROS, reactive oxygen species; SRF, serum response factor; AKAP, A kinase anchor protein. Bars, 10 µm.
Figure 4.

Phosphoproteomics analysis of cells invading HDFC matrix reveals complex downstream signaling. (A) Comparison of whole-genome microarray expression profiles for MDA-MB-231 cells with or without serum, or invading gelatin versus HDFC matrices, based on data pooled from five independent experiments for each condition. (B) Localization of activated β1 integrin to invadopodia of the MDA-MB-231 adherent to HDFC at the absence of serum. Invadopodia are yellow dots with colocalized actin and cortactin. (C) Localization of activated β1 integrin to FAs of MDA-MB-231 cells adherent to gelatin matrix in the absence of serum, showing poor invadopodia formation. (D) Comparative analysis of protein expression levels in MDA-MB-231 cells on HDFC versus cells on gelatin at the absence of serum. Cell lysates from each sample were labeled with specific iTRAQ labels to compare amounts of each protein (protein rank) identified by MS between HDFC and gelatin samples. (E) Numbers of unique and shared phosphoproteins and phosphopeptides in MDA-MB-231 carcinoma cells invading HDFC (green) compared with 2D gelatin (red) matrices in serum-free medium as identified by phosphoproteomics. (F) Quantification of types of phosphorylation sites identified by phosphoproteomics analysis. (G) Proposed signaling network associated with integrin-dependent induction of abundant invadopodia in carcinoma cells invading HDFC matrix. All identified phosphoproteins and their phosphosites are listed in Table S1. Open boxes indicate phosphoproteins identified in both HDFC and gelatin samples with the same phosphorylation sites by comparison of phosphopeptides. Gray boxes denote phosphoproteins identified in HDFC versus gelatin samples with different phosphorylation sites. Orange boxes indicate phosphoproteins unique to the HDFC matrix. Proteins without phosphorylation changes, such as Rac1, Cdc42, RhoA, and αβ integrin, are added to clarify the signaling context of proteins identified by phosphoproteomics. Although not regulated by phosphorylation, migfilin is depicted in the network because it is known to bind directly to both kindlin2 and filamin A. Migfilin and the other proteins highlighted by thick green outlines were verified experimentally in this study to play a role in invadopodia regulation by siRNA knockdown. Solid lines indicate known direct physical binding between proteins. Dashed lines indicate indirect interactions involving intermediate partners. Black arrows at the ends of lines indicate proteins known to stimulate the downstream signaling partner, lines with inhibition symbol indicate down-regulation of activity of the downstream signaling partner, lines with an inhibition symbol plus a black arrow indicate both potential activation and inhibition, and open arrows denote stimulation of the cellular process. Abbreviations used in this figure: ROCK, Rho-associated protein kinase; MLCP, myosin light-chain phosphatase; MLCK, myosin light-chain kinase; MPRIP, myosin phosphatase Rho-interacting protein; ROS, reactive oxygen species; SRF, serum response factor; AKAP, A kinase anchor protein. Bars, 10 µm.

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