Activation of the nonreceptor protein tyrosine kinase Ack by multiple extracellular stimuli

Abstract

Ack/Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile alpha motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.

Fig. 1.

Expression of Ack mRNA and protein. (A) Tissue distribution of Ack mRNA expression. A CLONTECH blot containing 2 μg of polyA+ RNA per lane was probed with a 5′ (Left) or a 3′ (Right) probe derived from the mouse Ack cDNA. In both cases, blots were exposed to film for 14 h at −80°C. (B) Characterization of Ack protein with site-specific anti-Ack antibodies. Mouse tissue homogenates were obtained from a 3-month-old male, and ≈60 μg of tissue lysates together with ≈20 μg of lysates of NIH 3T3 cells were analyzed with anti-Ack antibodies (Ab 880; see Fig. 6). (C) Constitutive tyrosine phosphorylation of Ack. 293 cells were transfected with either vector alone or vector containing full-length Ack in pRK5, and the recombinant protein was immunoprecipitated with antibodies against the amino terminus (Ab 729) or carboxyl terminus (Ab 730) of Ack and visualized with anti-Ack antibodies (Ab 880). Endogenous Ack from NIH 3T3 lysates was also analyzed in the same way by immunoprecipitation followed by immunoblotting. The blot was stripped and probed with anti-pTyr antibodies.

Fig. 2.

Integrin-mediated and growth factor-induced Ack activation. (A) Ack is activated upon cell attachment to fibronectin. NIH 3T3 cells were starved in serum-free medium (DMEM) for 20 h, and the cells were trypsinized and replated on fibronectin (FN)-coated 10-cm dishes. Cells were lysed, and extracts (0.5 mg) were immunoprecipitated with a combination of anti-Ack antibodies (Ab 729 and 730). Upon SDS/PAGE and transfer to nitrocellulose, immunoprecipitates were analyzed with anti-pTyr antibodies, then stripped and probed with anti-Ack antibodies (Ab 880). The blot was stripped again and probed with affinity-purified antibodies that specifically recognize tyrosine-phosphorylated Y284, an autophosphorylation site in the Ack kinase domain. (B) Integrin-mediated cellular redistribution of Ack. HeLa cells were transfected with an expression vector for Ack in pRK5. After 48 h, the cells were trypsinized and replated onto fibronectin-coated coverslips for the indicated time. At those times, nonattached cells were washed off, and attached cells were fixed with 4% paraformaldehyde, permeabilized, and stained with antibodies against pY284 at 1 μg/ml. The numbers below the pictures represent the percentage of stained cells on the coverslips. (C) Ack is activated and recruited by activated EGFR. Starved Her14 cells were stimulated with EGF for 5 min. Ack was immunoprecipitated from precleared lysates by using a combination of anti-Ack antibodies (Ab 729 and 730). Upon SDS/PAGE and transfer to nitrocellulose, the blot was probed with anti-pTyr antibodies. The blot was stripped and probed with anti-Ack antibodies (Ab 880) to visualize Ack and stripped again and probed with antibodies that recognize activated Ack (pY284). (D) Subcellular fractionation of Her14 cell lysates. Her14 cells were mock-stimulated (−) or EGF-stimulated (E) as in A and lysed in hypotonic lysis buffer (34). Upon separation of both soluble (Sol.) and particulate (Part.) fractions, equal amounts of protein (1 mg) were incubated with anti-Ack antibodies (Ab 730). Upon SDS/PAGE and transfer to nitrocellulose, immunocomplexes were analyzed with anti-pTyr antibodies. The blot was stripped and probed with anti-Ack antibodies (Ab 880). The blot was stripped again and probed with antibodies against pY284. (E) Ack is activated after PDGF or insulin stimulation. Starved L6 myoblasts were either mock-stimulated or stimulated with PDGF (P) or insulin (I) for 5 min. Ack was immunoprecipitated from the precleared lysates (≈0.3 mg) by using Ab 730, and immunocomplexes were analyzed with anti-pTyr antibodies. The blot was stripped and probed with anti-Ack (Ab 880) antibodies.

Fig. 3.

Endogenous Ack forms a stable complex with the adaptor protein Nck. (A) Her14 cells were starved overnight in DMEM with 0.1% calf serum and then either mock-stimulated or stimulated with EGF (E) for 5 min. Precleared lysates (≈2 mg) were incubated with anti-Ack (Ab 729) or anti-Nck antibodies previously crosslinked to protein A Sepharose beads. Immunocomplexes were analyzed with anti-Ack (Ab 730) or anti-Nck antibodies. (B) 293 cells were transfected with expression vectors for Nck and wild-type or mutant Ack as shown. (Left) Precleared lysates (250 μg) were immunoprecipitated with anti-Nck antibodies and analyzed by immunoblotting with anti-Ack (Ab 880) or anti-Nck antibodies. (Right) Whole-cell lysates (WCL; 30 μg) were directly analyzed by immunoblotting with anti-Ack (Ab 880) or anti-Nck antibodies.

Fig. 4.

Ack contains a SAM domain-like membrane-targeting region. (A) Alignment of the amino terminus of Ack (residues 1–85) to a panel of SAM domains in closely related protein kinases by using the program pfam. Secondary structure elements of this region consisting of five α-helixes were predicted by using psi-pred. (B) Cos1 cells were transfected with expression vectors for Ack-GFP (Left) or for SAM-like (N100) domain-GFP (Right). Cellular localization of fluorescently labeled Ack or SAM-like domain were visualized with an Axioskop2 microscope (Zeiss).

Fig. 5.

Role of Ack domains in regulation of Ack kinase activity. (A) HeLa cells were transfected with the indicated Ack wild type or mutants in the pRK5 expression vector. Forty-eight hours after transfection, cells were lysed and subjected to immunoprecipitation with anti-Ack antibodies that recognize wild type and Ack mutants (Ab 730). Tyrosine phosphorylation was detected by immunoblotting with anti-pTyr antibodies. The blot was stripped and probed with anti-Ack antibodies (Ab 880) to visualize the amount of Ack protein per lane. The blot was stripped again and probed with antibodies directed against the activated form of Ack (anti-pY284). (B) Cell extracts (≈0.4 mg) were incubated with 15 μg of purified GST–Ack SH3 fusion protein. After SDS/PAGE and transfer to nitrocellulose, bound proteins were analyzed by immunoblotting with anti-Ack (Ab 880) antibodies.