Purification of Nck binding proteins. (A) Detection of binding proteins from brain (B) and testis (Te) extracts. Total cytosolic extracts were passed through columns containing immobilized GST or GST-Nck SH3[2] as indicated. Proteins eluted with SDS treatment (left) were analyzed by GST–Ras-Nck SH3[2] binding. Differences between brain and testis samples are the p140 brain and the testis p110 binders. Numbers on the left are the molecular masses (in kilodaltons) of the markers, and those on the right indicate the positions of the six Nck binding proteins. (B) Sepharose Q ion-exchange fractionation of brain proteins was carried out as described in Materials and Methods. T, total extract; FT, flowthrough fraction; W, wash fraction; HS, high-salt fraction. The Nck SH3[2] binding proteins were assayed using 20 μl of material from each fraction. (C) Identification of Nck binding proteins by protein microsequencing. Bands were excised from the Coomassie blue-stained gels as shown and processed for tryptic digestion, high-performance liquid chromatography separation, and Edman microsequencing. At least three peptides with unambiguous sequences were used to identify the proteins as shown.

Biacore analysis of the interaction of SH3 domains with target sequences. (A) Superdex200 gel filtration analysis of GST-SrcSH3. The thrombin-cleaved material (dotted line) was treated with 10 U of thrombin per ml for 1 h at room temperature to release free SH3. The GST proteins are present exclusively as dimers, while MBP-Nck SH3[2] exhibits retention corresponding to a monomeric ∼50-kDa protein. Analytical gel filtration was conducted on the Smart system (Pharmacia) in HBS at 30 μl/min at 20°C. Standard protein markers (Sigma) are indicated by arrowheads: lysozyme, 14 kDa; carbonic anhydrase, 29 kDa; serum albumin, 66 kDa; and alcohol dehydrogenase 150 kDa. (B) Coupling (rU values) to CM5 channels was as follows: control channel GST, 2,110; GST-PAK18, 1,650; GST-PAK1-250, 1,760; and GST-PAKL404S, 2,280. The sensorgram shows the addition of 1 μM MBP-Nck SH3[2] at a flow rate of 10 μl/min.

Phosphorylation of PAK regulates Nck binding. (A) Effect of Cdc42-mediated activation of PAK on Nck SH3[2] binding. The top gel shows Coomassie blue-stained COS-7-expressed Flag-αPAK immunoprecipitated and incubated (30 min; 30°C) in the presence (+) of 2 μg of recombinant Cdc42 with (+) 0.1 mM GTPγS or GDP and/or 0.2 mM ATP as indicated. Neither Cdc42-activated αPAK nor intrinsically active recombinant GST-αPAKL107F binds Nck. As a control, the unphosphorylated GST-αPAKL404S (1 μg) bound normally. (B) PAK activation in vivo leads to dissociation of αPAK and Nck. COS-7 cells were transfected with 3 μg of the constructs per 100-mm-diameter plate as shown. The expression of αPAK is shown in the lower gel. Flag-Nck immunoprecipitates contain αPAK as expected, but not when the kinase is activated. As a control, kinase-dead (KD) αPAK(K298A) was found to be equally associated with Nck even with Cdc42^G12V present. Wt, wild type. (C) Phosphorylation of S21 blocks Nck binding. Increasing concentrations (micromolar) of synthetic peptides corresponding to PAK^9-23 or its phospho-S21 counterpart were coincubated with the Nck-SH3[2] probe. Each lane of the upper gel corresponds to a strip of filter incubated separately with the probe. The phosphorylated variant was unable to compete for binding to the GST-PAK^1-250 target (0.5 μg per lane).

PAK undergoes dynamic regulation of its interactions. PAK is capable of forming complexes with Nck and PIX through distinct proline-rich sequences. The kinase can be recruited to juxtamembrane sites either through the interaction of Nck with various tyrosine-phosphorylated receptors or by the interaction of PIX with elements of FCs. Activation of PAK requires autophosphorylation and causes FC disassembly; however, the kinase also loses affinity for its partners, resulting in PAK cycling back to the cytosol.

Protein overlays define multiple target proteins for certain SH3 domains. (A) Schematic diagram of the GST-Ras construct showing the arrangements of structural domains. [γ-³²P]GTP (asterisk) is introduced into the fusion protein by incubating the protein for 4 min in buffer containing 2.5 mM EDTA. Binding and washes are performed in buffers containing 5 mM MgCl₂. (B) Labeled SH3 domains as shown were overlaid onto blots (on PVDF) containing 80 μg of soluble proteins derived from rat tissues: brain (B), testis (Te), and heart (H). The filters were exposed for 4 h at −70°C. As a control, GST-Ras alone produces no signals. In addition to dynamin (∼100-kDa band in brain), the αPIX SH3 domain detects the three isoforms of PAK (α, β, and γ), with the level of αPAK being highest.

Identification of the Nck binding region in NIK and PAK. (A) Peptide sequences used to test Nck SH3[2] binding as indicated were expressed as GST fusion proteins. Residues conserved between PAK and NIK are in boldface; the arrowheads indicate the position of phosphorylated serine in PAK or nontolerated residues in NIK18 1. There is a phosphorylated serine in the Plx binding site (asterisk). (B) GST proteins displaying peptides (1 or 2) derived from PAK and NIK in panel A were analyzed by Nck SH3[2] overlay following separation of 2 μg of each protein on a 10% polyacrylamide gel. (C) PAK18 binds with efficiency similar to that of full-length PAK. A comparison of binding to 2 μg of αPAK(L404S) or 1 μg of the various peptides indicated in panel A is shown. The smaller peptide, PAK13, binds more weakly than PAK18; in this context, PAK13S21E fails to bind Nck. Numbers on the left indicate the molecular masses (in kilodaltons) of the markers.

Positional residue preferences for peptide binding to Nck SH3[2]. (A) Synthetic peptides corresponding to the αPAK Nck binding sequence (indicated on the left and marked by white crosses) and containing all possible point substitutions (260 peptides) were synthesized in an array. The filter was blocked with 1% bovine serum albumin and probed with ³²P-labeled Nck SH3[2] under standard conditions. A 10-min exposure is shown. (B) From phosphorimager analysis of panel A, preferred (highest counts) or required (boldface) and poorly tolerated (<20% of wild type) peptides are tabulated. Note that the presence of proline or acid residues in the C-terminal half of the peptide drastically reduces binding. (C) Identification of putative binding sequences in known Nck targets. Mammalian proteins identified by PHI-Blast (58) using the PAK 13 sequence and the motif P-X-[AIKLPRTV]-P-X-R-[exclude DEP]-[exclude DEP]-S. Matches were found in all targets that had previously been shown to directly bind Nck (asterisks), while other proteins with the potential to bind Nck are also shown. The conserved residues are highlighted in boldface. (D) Nck SH3[2] can bind to the proline-rich region of PTP-PEST (328 to 344); as a positive control, the NIK18 peptide (2 μg) was corun on a 10% polyacrylamide gel and analyzed by overlay for binding). Asterisks represent sites where only 1 residue is tolerated.

Identifying Nck and PAK interactions that are negatively regulated by phosphorylation. (A) Nck SH3[2] binding proteins detected in total proteins (80 μg/lane) from cultured cells are compared to those in brain extract. The sizes of the various targets are indicated; of these, p160 is suggested to represent NIK and p68 is suggested to represent αPAK. The identities of the relatively abundant p110 and p50 species are unclear. The right gel shows reduced Nck SH3[2] binding to HeLa cell extracts following 100 nM okadaic acid treatment (30 min) to induce general phosphorylation. Proteins with mobilities corresponding to 110 and 68 kDa showed no change; however, the p160, p60, and p58 bands showed significant loss of binding. These data are representative of two experiments. (B) Immunoprecipitated αPAK was activated in vitro with GTPγS-Cdc42 and probed with Ras-PIX SH3. Loss of binding correlated with autophosphorylation. The GST-αPAK^175-206 PIX-binding peptide (2 μg) was incubated with active recombinant αPAK (0.4 μg) plus 0.5 mM ATP (1 h; 30°C). The complete phosphorylation of S198 and S203 is indicated by its shifted mobility (phosphoprotein [pp]). The PIX SH3 overlay shows a decrease in binding due to this phosphorylation. (C) PAK binding to PIX-GIT1 is negatively regulated by kinase activation. COS-7 cells were transfected with combinations of expression plasmids as shown. The right gels show each of the proteins detected in total lysates (80 μg); the left gels show proteins associating with the expressed GST-GIT1. αPAK activation by Cdc42^G12V is accompanied by a mobility shift and loss of binding. +, present; −, absent.

Regulation of PAK association with FCs. (A) Inhibition of PAK leads to an accumulation of PAK in focal adhesions. NIH 3T3 cells were transfected with KID PAK^83-149 and stained with anti-αPAK and anti-vinculin. Only in cells expressing the inhibitor was there clear colocalization of PAK with the underlying focal adhesions. The inactive KID(L107F) mutant (KIDm) was used as a control. (B) HeLa cells were transfected (stars) as follows: Flag-αPAK (image 1), Flag-αPAK plus GST-KID (59) (image 2), and Flag-ΔN22αPAK plus GST-KID (image 3). Immunofluorescent detection of rabbit anti-Flag (Upstate Biotechnology Inc.) and mouse anti-vinculin was then performed. With inhibitor, the αPAK robustly stained vinculin-rich FCs, but PAK lacking the first 22 residues (i.e., the Nck binding site) was poorly localized to these structures. Bar, 10 μm.