Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration

Abstract

Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

Fig. 1.

PKA subunits and activity are enriched in Pd. (A) REF52 or WI38 fibroblasts were plated onto fibronectin-coated coverslips for 90 min, then stimulated for 1 h with 10 ng/ml PDGF (REF52) (Top and Middle) or 100 ng/ml LPA (WI38) (Bottom). Cells were fixed and processed for immunofluorescence by using antibodies against the indicated proteins and fluorescent phalloidin to stain F-actin as indicated, then examined by confocal microscopy. (Middle) Enlargements are shown of the area indicated by the square in Upper Left. (Scale bar: 10 μm.) (B) REF52 cells were cultured for pseudopod formation as described in Experimental Methods, then fixed, stained with Alexa 488-phalloidin, and imaged by confocal microscopy. (Left) The diagram shows the preparation and orients the reader to Center, which shows a 3D reconstruction of a single cell prepared and imaged as described. The top of the cell appears flat because the image stack stops approximately halfway through the cell nucleus. (Right) The same cell is shown, rotated upward ≈30° about the horizontal axis to illustrate the Pd's fine structure. Movie 1, which is published as supporting information on the PNAS web site, shows a 180° rotation of this image. (C and D) Twenty micrograms of protein prepared from CB from unstimulated cells (Un), or from CB and Pd formed in response to PDGF, EGF, and PDGF (10 ng/ml each; E+P), or LPA, were immunoblotted with the indicated antibodies. (E) PKA activity was determined from equal amounts of protein prepared from unstimulated (Ctrl) REF52 cells or CB (open bars) and Pd (filled bars) formed in response to LPA or PDGF. Data (as cpm per μg of lysate) represent means ± SD for three independent Pd preparations processed simultaneously for kinase activity.

Fig. 2.

Localized PKA activity correlates with enrichment of PKA-phosphorylated VASP. (A) REF52 CB and Pd, formed in response to PDGF, EGF, or LPA, were blotted for PKA-phosphorylated VASP (pVASP; p157-VASP) or the retinoblastoma protein (Rb), which resides in the nucleus and is therefore present only in CB. (B) CB from unstimulated NIH 3T3 cells (Un), or CB and Pd formed in response to PDGF or EGF, were blotted with an antibody against VASP. (C) COS7 cells transfected with epitope-tagged VASP (VSV-VASP) were either treated with 25 μM Fsk for 20 min or cultured for EGF-induced pseudopod formation. Lysates from Fsk-treated cells (Fsk) and from CB and Pd were immunoprecipitated with antivesicular stomatitis virus antibody. Precipitates and whole cell extract (wce) from transfected cells were separated by SDS/PAGE and blotted with the indicated antibodies. A low-percentage gel was used to collapse the phosphorylation-sensitive electrophoretic profile of VASP (evident in B) to a single band for easier confirmation of equal loading. Immunoblotting unprecipitated CB and Pd lysates with anti-Abl antibodies confirmed the presence of c-Abl in both fractions (Lower).

Fig. 3.

Localized regulation of PTP-PEST by PKA. (A and B) PTP-PEST was immunoprecipitated from extracts of REF52 cells stably adherent to tissue culture plastic (TC), CB, and Pd formed in response to PDGF, or Pd treated with mPKI or StHt31 (as described in Fig. 2), then separated by SDS/PAGE and blotted with anti-PTP-PEST and anti-phospho-PKA substrate (p-PKA sub) antibodies. (C) PTP-PEST was immunoprecipitated from PDGF-stimulated Pd (Ctrl), or Pd treated with mPKI or StHt31 as described above, and subjected to an in vitro phosphatase assay (see Experimental Methods) in which absorbance at 630 nm is proportional to released phosphate and, thus, phosphatase activity. Data represent means ± SD for three independent Pd preparations immunoprecipitated and processed simultaneously for phosphatase activity.

Fig. 4.

PKA activity controls Rac by regulating Rac GEF and Rac GAP activities within Pd. (A and B) NIH 3T3 cells were cultured as in Fig. 2 D and E and CB and Pd extracts were subject to a pulldown assay by using a GST-p21-binding domain fusion protein to isolate the active form of Rac. A portion of the extracts were collected before pulldown and immunoblotted directly to determine total Rac levels. The bar graphs depict the average ratios of active to total Rac, ± SD, determined from three separate experiments by densitometry of the immunoblotted bands. (C and D) Control- or inhibitor-treated Pd from PDGF-stimulated NIH 3T3 cells were harvested and incubated with purified, recombinant GST-Rac1 loaded with α-³²P-GTP (C) or γ-³²P-GTP (D) for the indicated times to measure Rac GEF or GAP activity, respectively. The data are presented as the percent of radioactivity remaining bound to Rac1 in the absence of extract and represent means ± SD for four independent Pd preparations processed simultaneously for GEF or GAP activity. Note that the y axis in C does not go to zero.

Fig. 5.

PKA activity and anchoring are required for pseudopod stability and formation and for chemotaxis. (A) REF52 cells were cultured for Pd formation toward PDGF for 1 h. PBS (Ctrl), 20 μM mPKI, or 50 μM StHt31 was added to the filter undersides and, at the indicated times, pseudopod formation was quantified by measuring the amount of pseudopod protein by using a bicinchoninic acid assay. Note that the y axis does not go to zero. (B and C) REF52 cells were cultured for Pd formation (B) or migration (C) by adding the indicated concentrations of StHt31, StHt31P, mPKI, cytochalasin D, or Fsk (in μM) to the cells 20 min before addition of growth factor. Pd formation was measured as above, whereas cell migration was measured as described in Experimental Methods. Similar results were seen by using NIH 3T3 cells (data not shown).