Individual Cas phosphorylation sites are dispensable for processive phosphorylation by Src and anchorage-independent cell growth

Abstract

Cas is a multidomain signaling protein that resides in focal adhesions. Cas possesses a large central substrate domain containing 15 repeats of the sequence YXXP, which are phosphorylated by Src. The phosphorylation sites are essential for the roles of Cas in cell migration and in regulation of the actin cytoskeleton. We showed previously that Src catalyzes the multisite phosphorylation of Cas via a processive mechanism. In this study, we created mutant forms of Cas to identify the determinants for processive phosphorylation. Mutants containing single or multiple YXXP mutations were phosphorylated processively by Src, suggesting that individual sites are dispensable. The results also suggest that there is no defined order to the Cas phosphorylation events. We also studied the effects of these mutations by reintroducing Cas into Cas-deficient fibroblasts. Mutants lacking some or all YXXP sites augment the ability of Src to promote anchorage-independent growth. On the other hand, deletion of YXXP sites compromises the ability of Cas to promote tumor cell migration.

FIGURE 1. Schematic representation of wild-type and mutant Cas proteins

A, domain structure of Cas showing the SH3 domain (SH3), proline-rich region (Pro), substrate region containing 15 repeats of YXXP, serine-rich region (Ser), C-terminal Src binding sequence (SBS), and helix-loop-helix region (HLH). B, diagram showing WT Cas and mutant forms of Cas used in this study with YDXP and YQXP motifs indicated above the diagram. In mutants Cas^F7, Cas^F12, and Cas^F17, 7, 12, and 17 tyrosine residues were mutated to Phe, respectively. Tyrosine residues in the substrate domain are numbered and shown in black, and mutated Tyr residues are shown in gray.

FIGURE 2. Single-site mutants of Cas are phosphorylated by Src

In vitro kinase assays were carried out at 30 °C using purified Cas proteins (WT or mutant), purified v-Src, and [γ-³²P]ATP. Aliquots were withdrawn at the indicated times and analyzed by SDS-PAGE and autoradiography. To quantify the rates of phosphorylation, densitometry was carried out on a Bio-Rad GS-710 imaging densitometer with QuantityOne software. Rates are presented on the right.

FIGURE 3. Phosphorylation of Cas multisite mutants

A, in vitro reactions were carried out using Cas proteins and 100 µm unlabeled ATP for 20 min in the presence or absence of v-Src. The reactions were analyzed by SDS-PAGE and silver staining. B, time courses for Cas phosphorylation were carried out using methods similar to those described in A. Aliquots were withdrawn at the indicated times and analyzed by SDS-PAGE and silver staining.

FIGURE 4. Kinetics of wild-type Cas phosphorylation at different substrate concentrations

A, WT Cas was incubated in the presence of 50 nm v-Src at 30 °C in kinase assay buffer. Reactions were started by the addition of 100 µm unlabeled ATP plus [γ-³²P]ATP (5 µCi). Aliquots were withdrawn at the indicated times and analyzed by 8% SDS-PAGE and autoradiography. B, incorporation of ³²P into Cas was measured by exposing the dried gels to a PhosphorImager screen (GE Healthcare) and quantifying the band intensity using ImageQuant software. A graph of velocity (PhosphorImager units/min) versus substrate concentration is shown. The data were fit to the Michaelis-Menten equation using nonlinear regression analysis.

FIGURE 5. Kinetics of mutant Cas phosphorylation at different substrate concentrations

A, Cas^F7 was incubated in the presence of 50 nm v-Src at 30 °C in kinase assay buffer. Reactions were started by the addition of 100 µm unlabeled ATP plus [γ-³²P]ATP (5 µCi). Aliquots were withdrawn at the indicated times and analyzed by 8% SDS-PAGE and autoradiography. B, incorporation of ³²P into Cas was measured by exposing the dried gels to a PhosphorImager screen and quantifying the band intensity using ImageQuant software. A graph of velocity (PhosphorImager units/min) versus substrate concentration is shown. The data were fit to the Michaelis-Menten equation using nonlinear regression analysis. C, Cas^F12 phosphorylation reactions were analyzed by SDS-PAGE as described above in A. D, Cas^F12 phosphorylation was quantified by PhosphorImager as shown in B.

FIGURE 6. Phosphorylation of WT and mutant forms of Cas at different Src concentrations

A, left, pulse-chase experiments were carried out on WT Cas at different Src concentrations. In the pulse reaction, Cas and Src were incubated on ice in the presence of 0.1 µm[γ-³²P]ATP. After 3 min, aliquots were removed from the reaction and diluted into four separate tubes containing kinase assay buffer and excess unlabeled ATP (0.5 mm final) to give final Src concentrations of 100, 200, 400, and 600 nm. The chase reaction was carried out at 30 °C for 25 min. Reactions were stopped by adding SDS-PAGE sample buffer and boiling. Equal amounts of Cas from each reaction were analyzed by SDS-PAGE and autoradiography. Aliquots removed after 3-min pulse reaction were also analyzed on the gels. Right, in separate pulse-chase reactions, time courses were followed at Src concentrations of 100 and 600 nm. B, similar experiments were performed for Cas^F7. C, similar experiments were performed for Cas^F12.

FIGURE 7. Add-back mutants of Cas are phosphorylated by Src

In vitro kinase assays were carried out at 30 °C using Cas proteins (WT or add-back mutants), v-Src, and [γ-³²P]ATP. Aliquots were withdrawn at the indicated times and analyzed by SDS-PAGE and autoradiography.A, phosphorylation of add-back mutant Cas^F17 + Tyr²⁵³. B, phosphorylation of add-back mutant Cas^F17 + Tyr²⁵³/Tyr¹⁹⁶.

FIGURE 8. Expression of WT and mutant Cas in Cas^−/− fibroblasts

Cas-deficient fibroblasts were transfected with wild-type or mutant forms of Cas (in pBABEhygro) and v-Src (in pBABEpuro) and selected for hygromycin and puromycin resistance. Cells marked minus were transfected with the appropriate vector controls. Cells were lysed in modified radioimmune precipitation assay buffer, and lysates (30 µg) were analyzed by immunoblotting using Cas, v-Src, or β-actin antibodies. A, Cas Y253F, Cas^F17, and Cas^F17 + Tyr²⁵³ mutants. B, Cas^F7 and Cas^F12 mutants.

FIGURE 9. Effects of Src and Cas on Src-mediated anchorage-independent growth and migration

A and B, anchorage-independent growth. 20,000 cells were plated in 12-well tissue culture plates. Cell numbers were obtained using a Coulter counter after 11 days. Open bars, cells without Src; closed bars, cells expressing Src. C and D, cell migration assays. 200,000 cells were plated on cell culture inserts (3-µm pore size) in 6-well plates. Cell migration was measured after 72 h as the percent of cells found in the bottom well/total cell number. E, cell migration assays were carried out using 8-µm pore size membranes, and cell migration was examined after 24 h.