Paxillin Serves as an ERK-Regulated Scaffold for Coordinating FAK and Rac Activation in Epithelial Morphogenesis  Shuta Ishibe, Dominique Joly, Zhen-Xiang Liu, Lloyd G. Cantley  Molecular Cell  Volume 16, Issue 2, Pages 257-267 (October 2004) DOI: 10.1016/j.molcel.2004.10.006

Figure 1 ERK Phosphorylates Paxillin on Serine 83 (A) In vitro incubation of gst-N-Paxillin ± active ERK followed by immunoblotting with α-pS83 (top) and α-paxillin (bottom). (B) Quiescent mIMCD-3 cells treated ± HGF (40 ng/ml) for 10 min followed by immunoblotting cell lysates as above. (C) mIMCD-3 cells were treated ± U0126 (10 μM for 20 min) prior to simulation ± HGF for 10 min. Paxillin was immunoprecipitated from cell lysates followed by immunoblotting as above. (D) mIMCD-3 cells were transiently transfected with either myc-tagged wild-type paxillin (myc.Pax) or myc-tagged S83A paxillin (myc.PaxS83A) constructs followed 48 hr later by treatment ± HGF. Lysates were immunoprecipitated with α-myc followed by immunoblotting as above. (E) Quiescent nonconfluent mIMCD-3 cells were treated with 40 ng/ml HGF for the indicated times and cell lysates immunoblotted with α-pS83 (top) and α-paxillin (bottom). (F) Quantification of phosphorylation at Ser-83 was performed by scanning densitometry of the bands and normalization to the total amount of paxillin present in the sample. Relative values shown are the means ± SEM (n = 3). Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 2 ERK-Phosphorylated Paxillin is Localized at Focal Adhesions (A) mIMCD-3 cells plated for 24 hr were stimulated ± HGF, fixed, and simultaneously immunostained for paxillin and pS83 paxillin. In the bottom panel, the pS83 paxillin antibody was pre-incubated in a ∼1000-fold molar excess of the immunizing phosphopeptide (competitive peptide, CP) prior to immunostaining. Arrows indicate superimposition of paxillin and pS83 paxillin. Asterisks indicate a nuclear signal detected with the pS83 antibody that does not colocalize with paxillin. (B) mIMCD-3 cell clones expressing myc-tagged wild-type paxillin (WT4) plated for 24 hr were stimulated ± HGF, fixed, and stained for myc and pS83 paxillin. Arrows indicate superimposition of myc and pS83 paxillin staining at focal adhesions. Arrowheads demonstrate weak detection of ERK-phosphorylated paxillin in paxillin overexpressing cells even in the absence of HGF treatment. (C) mIMCD-3 cells expressing S83A paxillin (clone S6) plated for 24 hr were treated as in (B). Arrows indicate localization of the myc-tagged paxillin to focal adhesions. Phosphorylation of paxillin by ERK is weakly detected in these cells (arrowhead), presumably due to low level expression of the endogenous protein (as seen in clone S30, Supplemental Figure S2). Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 3 Paxillin Phosphorylation Regulates FAK Association (A) Cells transiently transfected with either myc-tagged wild-type, S83A, or 118ASVA paxillin were treated ± HGF followed by immunoprecipitation with α-myc and immunoblotting with α-pERK and α-paxillin. (B) Cells transiently transfected as above were treated ± HGF followed by immunoprecipitation with α-myc and immunoblotting with α-pS83 and α-paxillin. (C) Subconfluent mIMCD-3 cells were treated ± U0126 for 20 min followed by stimulation ± HGF for 10 min. Cell lysates were then immunoprecipitated with α-paxillin, and associated proteins separated by SDS-PAGE and immunoblotted with α-FAK and α-paxillin. (D) Lysates of subconfluent mIMCD-3 cells stimulated ± HGF for 10 min were immunoprecipitated with α-pS83 and the immunoprecipitate blotted with α-FAK. Whole cell lysates were immunoblotted with α-paxillin to judge equality of starting material. (E) Subconfluent mIMCD-3 cells transiently transfected with either myc-tagged wild-type, S83A, or 118ASVA paxillin were treated ± HGF for 10 min followed by immunoprecipitation with α-myc and immunoblotting with α-FAK and α-myc. (F) Densitometric quantification of five independent experiments was performed as in (E). *p < 0.01 versus no HGF. (G) Subconfluent mIMCD-3 cells were treated as in (E) followed by immunoblotting with a phosphospecific antibody to tyrosine 397 in FAK (α-pFAK) and α-myc (to judge equality of paxillin immunoprecipitation). (H) Densitometric quantification of three independent experiments was performed as in (G). *p < 0.01 versus no HGF. Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 4 FAK Recruitment to Paxillin Is Required for PI 3-K and Rac Activation (A) Cells stably expressing either myc.Pax (wt), myc.PaxS83A, or myc.PaxASVA were treated ± HGF followed by immunoprecipitation with α-FAK and immunoblotting with α-p85 and α-FAK. (B) Densitometric quantification of 3 independent experiments from two separate clones was performed as in (A). *p = 0.04 versus no HGF. (C) Cells as in (A) were treated ± HGF and whole-cell lysates immunoblotted with α-pAkt (an antibody that recognizes the activated form of Akt) and α-Akt. (D) Densitometric quantification of five independent experiments from two separate clones was performed as in (C). *p = 0.02 versus HGF treatment in wild-type cells; **p = 0.04 versus HGF treatment in wild-type cells. (E) Cells as in (A) were treated ± HGF followed by PBD pulldown of GTP-Rac and immunoblotting with α-Rac. Whole-cell lysates were immunoblotted with α-Rac (detecting both GDP-Rac and GTP-Rac) to determine equality of starting material. (F) Densitometric quantification of 3 independent experiments from two separate clones was performed as in (E). *p < 0.01 versus no HGF. (G) mIMCD-3 cells were pretreated for 20 min with either U0126 (10 μM) or LY294002 (50 μM), followed by stimulation with HGF (40 ng/ml, 10 min) and PBD pull-down of GTP-Rac performed as in (E). (H) Either PI4,5P2 (PIP2, 10μM) or PI3,4,5P3 (PIP3, 10μM) was added to serum-starved mIMCD-3 cells for 10 min, followed by cell lysis and PBD pulldown of GTP-Rac as in (E) and (G). Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 5 FAK Colocalizes with ERK-Phosphorylated Paxillin at Focal Adhesions (A) Quiescent mIMCD-3 cells plated for 24 hr were treated ± HGF, fixed, and immunostained for FAK and pS83 paxillin. Arrows indicate FAK and phospho S83 paxillin colocalizing at focal adhesions after HGF stimulation. Arrowheads indicate FAK localization in vehicle treated cells (upper panels). (B) mIMCD-3 cell clones expressing high levels of myc-tagged wild-type paxillin (WT17), high levels of the S83A paxillin mutant (S30), or low levels of the S83A paxillin mutant (S20) were plated on fibronectin coated culture slides for 30 min in the presence of HGF, fixed, and immunostained for myc (to detect the myc-tagged paxillin) and FAK. Arrows indicate colocalization of wild-type paxillin and FAK at the base of extending lamellipodia. Arrowheads demonstrate colocalization of FAK and paxillin S83A in lamellipodia of the low-expressing cells. Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 6 Mutation of Serine 83 Prevents HGF Stimulated Cell Morphogenesis (A) Stable mIMCD-3 clones expressing myc-tagged wild-type paxillin (WT4), S83A (S6), or 118ASVA (A11) were seeded on 24-well plates ± HGF for 180 min, and representative individual cells photographed using a Nikon microscope with Hoffman modulation (40×). (B) Quantitation of the relative surface area of cells as in (A) was determined by using the NIH Image software. Data represent mean ± SEM from results of two separate clones for each condition. *p < 0.01 versus no HGF. (C) Stable mIMCD-3 clones expressing myc-tagged wild-type paxillin (WT16), S83A (S10), or 118ASVA (A6) were assayed for single cell migration as described and representative fields photographed (20×). Cell processes extending through membrane pores are indicated by the arrows; open membrane pores are indicated by the arrowheads. Cell migration was only scored positive if nuclei were visible (*). (D) Quantification of cell migration from four separate experiments by using two separate clones for each condition. *p < 0.01 versus no HGF. (E) mIMCD-3 cell clones expressing myc-tagged wild-type paxillin (WT17), S83A (S6), or 118ASVA (A11) were incubated in type 1 collagen gels ± HGF for 24 hr, and representative single cells photographed for each condition (40×). (F) The number of processes/cell was determined from 30 cells/well treated as in (E), scored in triplicate wells from three separate experiments by using two separate clones for each condition. *p < 0.01 versus no HGF. (G–L) mIMCD-3 cell clones expressing the myc-tagged paxillin constructs were incubated in a Matrigel/collagen mixture ± HGF for 7 days. Representative structures were photographed on day 7 at 20× (G, I, K) and 40× (H, J, L). G and H) Cells expressing wild-type paxillin formed extensive multicellular branching tubules with lumens (WT17, arrows). (I and J) Cells expressing the S83A (S30) paxillin mutant formed primarily cystic structures in response to HGF (asterisk), with many cysts containing cells that extended short processes from the outer edge of the cyst (arrowheads). (K and L) Cells expressing the 118ASVA (A6) mutant were indistinguishable from cells expressing S83A paxillin. Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)

Figure 7 A Model of HGF-Stimulated Paxillin-MAPK Signaling in Focal Adhesions Paxillin, by serving as a local scaffold for ERK recruitment and activation, is itself a substrate for ERK phosphorylation, resulting in the regulated association of paxillin and FAK and the local activation of the PI 3-K. By using this signaling scaffold, both the activation of Rac and the FAK-dependent turnover of existing focal adhesions can be spatially regulated in the vicinity of the activated c-met receptor. Molecular Cell 2004 16, 257-267DOI: (10.1016/j.molcel.2004.10.006)