PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR

Abstract

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E(3)-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin -ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Figure 1

Cytoskeletal changes induced by long-term stimulation with PDGF, IGF-1, EGF and their combinations. Swiss 3T3 cells were stimulated with growth factors for 18 h and stained with α-vinculin antibody and rhodamine–phalloidin. Arrowheads and arrows show FAs and dendritic protrusions, respectively. Note the dominance of PDGF in costimulation experiments, resulting in an elongated cell shape with dendritic protrusions and disruption of SFs and FAs.

Figure 2

PDGF-specific suppression of MRLC. (A) Abundance of radiolabelled MRLC in growth factor-treated Swiss 3T3 fibroblasts at 6, 12 and 18 h (encircled in black). Protein synthesis was studied by labelling with [³⁵S]-methionine and -cysteine during the last 3 h of stimulation. (B) Changes in the abundance of nascent acidic MRLC induced by PDGF, IGF-1 and EGF (left side of graphs), and the effects of costimulation for 18 h (right). Quantitative data represent average of three independent analyses of cells at different times. (C) Semiquantitative RT–PCR analysis of MRLC 2A gene expression in response to growth factor stimulation. (D) PDGF treatment dominantly suppressed the levels of nascent MRLC in costimulation experiments at 18 h. The acidic isoform is encircled in white, and indicated by horizontal arrow pointing left. (E) Effect of various inhibitors on the acidic MRLC isoform in cells exposed to PDGF for 18 h. DMSO (1 μl/ml), LY294002, rapamycin, PD98059 or PS-341 in DMSO was added 1 h before and the 3 h labelling period. (F) Immunoblots of MRLC after GF stimulation. Staining was performed with anti-MLC and β-actin antibodies from Sigma. (G) MRLC levels in controls and in cells stimulated for 4 h with PDGF in the presence of cycloheximide (CHX) (10 μg/ml) or CHX plus lactacystin (50 μM). (H) Changes in MRLC expression after CHX-chase. Cells were stimulated with GF for 1 h before CHX. Stained with anti-MLC and anti β-actin antibodies as above. (I) Localization of MRLC. Growth factor-treated 3T3 cells were stained with anti-MLC antibody (1:25; Sigma) and rhodamine–phalloidin for visualization of actin. Colocalization with actin SFs is indicated by arrowheads in left images and local concentrations at the termination of the PDGF-specific surface extensions are indicated by arrowheads to the right.

Figure 3

PDGF-specific ubiquitination of MRLC in 3T3 fibroblasts. (A) Cells were treated with PDGF for 18 h and 150 μg of proteins separated by 2DE. MRLC was detected by immunoblotting with a murine IgM mAb (1:200; NEB). The 22 kDa isoform migrating at a pI of 4.55 is encircled in black, and indicated by horizontal arrow at the right. (B) Controls- (C) and PDGF- (P) treated cells were lysed and MRLC isolated by immunoprecipitation (1:25; sc-9448). Immunostaining with anti-ubiquitin antibody (1:1000; Affiniti Research Products, PW8810) demonstrated increased ubiquitination of MRLC in treated cells. The poly-ubiquitin-MRLC form migrated at a molecular weight of approximately 150 kDa, similar to the size of the HMW immunoreactive species indicated by oblique upwards arrows in (A). (C) Polyubiquitinated MRLC was increased by PDGF stimulation for 1 h in the presence of 1 μM PS-341, compared with that in EGF- or IGF-1-treated cells. Costimulation with PDGF also enhanced MRLC ubiquitination. The anti-ubiquitin-stained membrane was subsequently stripped and reprobed with antibody against MRLC (1:200; NEB) (bottom row). (D) The membrane was restained to show clearly the presence of ubiquitinylated MRLC at 150 kDa. (E) The 150 kDa polyubiquitinated MRLC was less abundant in IGF-1- than in PDGF-stimulated cells.

Figure 4

Induction of the MIR E3-ligase by PDGF is abrogated by Src-kinase inhibition. (A) Increase in the 50 kDa MIR ubiquitin-ligase after GF stimulation. Rabbit anti-MIR antiserum was used at 1:3000. The levels were increased at 15 min, with a maximum attained at 1 h after PDGF stimulation. (B) Effects of DMSO, EGF, PDGF or PDGF plus 2 μM SU6656 (all in DMSO) on MIR. (C) Copurification of MIR and phosphorylated MRLC. Cells were labelled with ³³P-ortho-phosphate for 6 h before PDGF stimulation and MIR isolated by immunoprecipitation. A 22 kDa phosphoprotein specifically copurified with MIR is indicated by oblique downward arrow in the autoradiogram (left). Subsequent staining of the membrane with the antibody specific for MRLC 2 phosphorylated at Thr18 and Ser19 (no. 3674; Cell Signaling) is shown to the right. (D) Inhibition of Src-kinase activity by SU6656 abolished the PDGF-specific increase in MRLC degradation. (E) Morphology of Swiss 3T3 cells after 18 h GF stimulation in the presence and absence of 2 μM SU6656. (F) 2D autoradiograms of phosphoisotope-labelled proteins immunoprecipitated with a monospecific antibody against hnRNP-K protein (enlarged gel area is shown). The direction of IEF and SDS–PAGE is indicated in the upper right corner, and in all images, the acidic side is to the left. A hnRNP-K phosphoform induced in response to all three GFs, but absent in untreated cells, is indicated by white downward arrows. The position of hnRNP-K was confirmed by immunoblotting (G) and is indicated in the image from EGF-treated cells. Note the increased hnRNP-K phosphorylation in PDGF-stimulated and -costimulated cells, and the appearance of new phosphoforms (oblique downward arrows in PDGF images). A copurified protein (indicated by a horizontal black arrow) migrating slightly lower than hnRNP-K and demonstrating increased phosphorylation in IGF-1- and PDGF-stimulated cells was identified as p85α using a mixture of the mAbs U9 and U14 (H). IGF-1- and PDGF- but not EGF-induced PI3 K activity in the 3T3 cells (see Supplementary Figure 2), and phospho-activation of hnRNP-K-associated p85 coincites with increased PI3 K activity in IGF-1- and PDGF-treated cells, further verifying the specificity of the phosphoisotope labeling procedure. (I) RT–PCR amplification of hnRNP-K associated MIR mRNA isolated by immuno precipitation from samples from nonstimulated and cells exposed to GF for 60 min using the monospecific antibody 54.

Figure 5

PDGF-induced morphology, migration and wound healing are significantly affected by silencing of MIR E₃-ligase. (A) Silencing of MIR by siRNA shown by Western blotting. Lane 1: control, no transfection; lane 2: transfection no siRNA; lane 3: scramble (scr) siRNA; lane 4: MIR siRNA 1; lane 5: MIR siRNA 2; lane 6: MIR siRNA 1+2. Note the additive effect with both siRNAs. (B) Morphological changes after silencing of MIR. Transfection with MIR-specific oligos but not with scramble ones abrogated the changes in morphology induced by PDGF. IGF-1-treated cells were unaffected. Note the nuclear localization of MIR in PDGF and its absence in IGF-1-treated cells (upward white arrows). (C) 3T3 cells with low MIR exhibited decreased migration after PDGF stimulation, as shown using the scratch assay (see Materials and Methods). Cells were photographed at time 0 and 18 h after a scratch. (D) Quantification of data. Note the significant reduced migratory potential of the siMIR-treated cells. Silencing of MIR had no effects on immotile IGF-1-treated fibroblasts. MIR downregulation and total cellular MRLC levels are shown by the immunoblots.

Figure 6

Inhibition of proteasome or Src-kinase activity prevent PDGF-induced differentiation of Swiss 3T3 cells and results in intranuclear accumulation of MRLC. (A) Growth factor induced changes in the localization of MRLC and actin demonstrated by staining with anti-MLC antibody (1:25; Sigma) and by rhodamine–phalloidin staining after 18 h stimulation. (B) Lactacystin (50 μM), inhibiting proteasomes, abolished PDGF-specific morphological changes, resulting in an increase in nuclear MRLC staining similar to that seen in untreated, and EGF- and IGF-1-treated cells. (C) Effects of lactacystin and SU6656 on the nuclear abundance of MRLC in PDGF-stimulated cells. Stained with anti-MLC antibody (1:100; Sigma). Lamin B1 served as nuclear marker. (D) Inhibition of Src-kinase activity by SU6656 abolished PDGF-specific morphologic changes and resulted in resulting in an increase in nuclear MRLC staining similar to that seen in untreated, and EGF- and IGF-1-treated cells. (E) MRLC in cytosolic fractions. Stained with the anti-MRLC 2 antibody (1:200; Santa-Cruz, sc-9448).