Inhibition of src family kinases by a combinatorial action of 5'-AMP and small heat shock proteins, identified from the adult heart

Abstract

Src family kinases are implicated in cellular proliferation and transformation. Terminally differentiated myocytes have lost the ability to proliferate, indicating the existence of a down-regulatory mechanism(s) for these mitogenic kinases. Here we show that feline cardiomyocyte lysate contains thermostable components that inhibit c-Src kinase in vitro. This inhibitory activity, present predominantly in heart tissue, involves two components acting combinatorially. After purification by sequential chromatography, one component was identified by mass and nuclear magnetic resonance spectroscopies as 5'-AMP, while the other was identified by peptide sequencing as a small heat shock protein (sHSP). 5'-AMP and to a lesser extent 5'-ADP inhibit c-Src when combined with either HSP-27 or HSP-32. Other HSPs, including alphaB-crystallin, HSP-70, and HSP-90, did not exhibit this effect. The inhibition, observed preferentially on Src family kinases and independent of the Src tyrosine phosphorylation state, occurs via a direct interaction of the c-Src catalytic domain with the inhibitory components. Our study indicates that sHSPs increase the affinity of 5'-AMP for the c-Src ATP binding site, thereby facilitating the inhibition. In vivo, elevation of ATP levels in the cardiomyocytes results in the tyrosine phosphorylation of cellular proteins including c-Src at the activatory site, and this effect is blocked when the 5'-AMP concentration is raised. Thus, this study reveals a novel role for sHSPs and 5'-AMP in the regulation of Src family kinases, presumably for the maintenance of the terminally differentiated state.

FIG. 1

Demonstration of c-Src inhibitory activity in the cardiocyte lysate. (A) c-Src autophosphorylation and enolase substrate phosphorylation measured in the absence (−) or presence (+) of the cardiocyte lysate (0.1 μg/μl). The positions of c-Src (60 kDa) and enolase (45 kDa) are indicated. (B) Dose response of the effects of cardiac lysate on c-Src activity determined with p34^cdc2 synthetic peptide as the substrate. (C) Activity and amount of c-Src in the kinase reaction determined by autoradiography and Western blotting (W. Blot) with c-Src monoclonal antibody GD11, respectively. (D) Cardiocyte lysate was split into <10- and >10-kDa fractions as described in Materials and Methods. Top, c-Src autophosphorylation in the absence or presence of cardiocyte lysate, the <10-kDa fraction, and/or the >10-kDa fraction; bottom, similar experiment performed with c-Src preautophosphorylated with [γ-³²P]ATP, where the effect of cardiocyte fractions was measured subsequently with unlabeled ATP. (E) The <10-kDa fraction was treated with buffer, subtilisin, or pronase and then used to study their effects on c-Src autophosphorylation in the absence or presence of the untreated >10-kDa (left) or <10-kDa (right) fraction.

FIG. 2

Inhibitory components are present predominantly in the heart tissue and preferentially inhibit Src family kinases. (A) Adult feline tissue samples were processed to obtain heat-stable lysates. The effect of each lysate (final concentration, 0.1 μg of protein/μl) on c-Src autophosphorylation was measured in the absence (top) or presence of cardiocyte <10-kDa fraction (final concentration, 0.16 U; middle) or >10-kDa fraction (final concentration, 0.1 μg of protein/μl; bottom). c-Src activity in the absence or presence of cardiocyte fractions is shown in the buffer lane. ³²P-labeled c-Src was detected by autoradiography. (B) Combinatorial effects of <10- and >10-kDa components on the autophosphorylation of c-Src, Lyn, Fyn, and EGF-R and MBP substrate phosphorylation by ERK-2 and p34^cdc2 kinase. The concentrations of <10- and >10-kDa fractions were determined as described in Materials and Methods. Left, effects of increasing amounts of the >10-kDa fraction in the presence of a constant amount of the <10-kDa fraction (final concentration, 0.16 U; 1 U = 3.0 AU at 259 nm); right, effects of increasing amounts of the <10-kDa fraction in the presence of a constant amount of the >10-kDa fraction (final concentration, 0.1 μg of protein/μl). Kinase activity in the absence of both cardiocyte fractions is shown in buffer lanes. c-Src autophosphorylation was measured at either 1 (uppermost panel) or 10 (lowermost panel) μM ATP. Autophosphorylation of other tyrosine kinases was determined at 1 μM ATP, whereas the MBP phosphorylation by p34^cdc2 and ERK-2 was measured at 10 μM ATP due to their higher K_m for ATP. The ³²P-labeled tyrosine kinases and MBP were detected by autoradiography. (C) Activities of c-Src and v-Abl measured in the absence or presence of either <10-kDa fraction (final concentration, 0.16 U), >10-kDa fraction (final concentration, 0.05 μg of protein/μl), or both. After autoradiography, the bands were cut for Cerenkov counting.

FIG. 3

The <10-kDa nonprotein component was purified and identified as 5′-AMP. (A) The <10-kDa cardiocyte fraction was chromatographed on a Pharmacia PepRPC column (graph); fractions (Fr.) showing inhibitory activity (fractions 9 and 10) on c-Src autophosphorylation in the presence of the >10-kDa fraction are shown in the autoradiograph. (B) PepRPC inhibitory fractions were pooled and chromatographed on a Pharmacia Superdex column (graph). A major peak fraction (fraction 42) exhibiting combinatorial inhibitory effect on c-Src autophosphorylation (autoradiograph) corresponds to a molecular mass of 350 Da. The column was precalibrated as described in Materials and Methods. (C) Molecular mass determination of the purified fraction from a Superdex column (top) and 5′-AMP with purity of >99% (bottom) was performed by ESI-mass spectroscopy. The molecular mass of the purified sample was determined to be 347.8 Da, matching that of 5′-AMP. (D) 1H NMR spectra of the purified fraction from the Superdex column (top) and 5′-AMP (bottom) were determined as described in Materials and Methods. In the NMR profiles, dimethyl sulfoxide (DMSO) and H₂O lines are marked, and an unmatched minor line in the purified sample is indicated by an asterisk. (E) Combinatorial c-Src inhibitory activity due to the addition of increasing amounts of either the purified Superdex column fraction (sample) or 5′-AMP, measured in the presence of cardiocyte >10-kDa fraction (0.1 μg/μl, final concentration). Nucleotide concentrations (Conc) in the purified <10-kDa fraction and in 5′-AMP samples were adjusted on the basis of absorbance at 259 nm. c-Src autophosphorylation was quantitated by Cerenkov counting (graph). (F) Inhibitory effect by the combined action of 5′-AMP and the >10-kDa fraction in assays using other nucleotide analogs. c-Src autophosphorylation was measured with increasing concentrations of AMP isomers and related nucleotides. In the absence (−) of the >10-kDa fraction, the effect on c-Src activity was measured with two higher concentrations (0.08 and 0.16 μg/μl) of nucleotides. ³²P-labeled c-Src was detected by autoradiography.

FIG. 4

Purification and identification of the >10-kDa component as an sHSP. (A) The >10-kDa fraction (∼1 mg), prepared as described in Materials and Methods, was chromatographed (data not shown) on an anion-exchange column (Mono Q; Pharmacia). Flowthrough fractions (Fr.) containing the inhibitory protein component were pooled, dialyzed, and chromatographed (graph) on a molecular sizing column (Superose 12; Pharmacia). Proteins eluted at an approximate molecular size of 18 kDa showed inhibition of c-Src autophosphorylation in the presence of cardiocyte <10-kDa fraction (bottom). (B) Purification of the >10-kDa protein component by sequential chromatography. Amounts of protein loaded to and eluted from each column are indicated in boxes and brackets, respectively. Differences in the amounts of protein recovered from an earlier column and the amounts of protein loaded to the next column are due to material saved for analyses and/or losses during sample processing. Buffer conditions for each chromatographic step and elution conditions (shown in parentheses) are indicated. Briefly, the detergent-free >10-kDa fraction (40 mg) was applied to a Superose 6 column equilibrated in sodium phosphate buffer. Inhibitory proteins eluted in the molecular mass range of ∼10 to 100 kDa were applied to a hydroxyapatite column equilibrated in sodium phosphate buffer. The inhibitory fractions were pooled and applied to a Pro-RPC column equilibrated in 0.1% TFA solution. Fractions demonstrating inhibitory activity were pooled and applied to a Cibacron blue column equilibrated in sodium phosphate buffer. The inhibitory fractions were pooled and used for protein sequencing. For details, see Materials and Methods. (C) Edman sequencing of part of the purified sample from Cibacron blue chromatography performed after digestion of the purified sample with CNBr. The sequence of the digested sample shows strong homology with that of αA-crystallin and matches the conserved residues (indicated in boxes) of other sHSP family members. (D) Effects of various HSP family members on c-Src autophosphorylation in the presence (+) or absence (−) of 5′-AMP compared with that of the cardiocyte >10-kDa fraction. c-Src autophosphorylation was measured with increasing amounts of either >10-kDa fraction or HSPs. In the absence of 5′-AMP, the direct effects of these proteins on c-Src autophosphorylation were determined with two higher concentrations (0.08 and 0.16 μg/μl). ³²P-labeled c-Src was detected by autoradiography.

FIG. 5

Mechanism of c-Src inhibition. (A) Various concentrations of ATP (1, 10, 100, and 1,000 μM) in the c-Src kinase reaction mixture were used to determine whether the 5′-AMP- or 5′-ADP-mediated combinatorial inhibitory activity on c-Src is competitive with ATP. The effects of these nucleotides (ADP and AMP) on c-Src autophosphorylation were analyzed either in the presence (+) or absence (−) of the cardiocyte >10-kDa fraction. c-Src autophosphorylation in the absence of nucleotides but in the presence or absence of the >10-kDa fraction is shown for controls. (B) Effect of c-Src inhibitory activity on the preautophosphorylated c-Src in cardiocyte lysate. A 15-min standard kinase assay was performed for c-Src autophosphorylation as well as α-casein substrate phosphorylation in the absence or presence of cardiocyte lysate. In part of the assay, performed in the absence of the cardiocyte lysate (last two lanes), we performed an additional 15-min kinase reaction after omitting or including the cardiocyte lysate. ³²P-labeled c-Src (top) and α-casein (doublet in the lower autoradiograph) were detected by autoradiography. (C) c-Src autophosphorylation time course determined under standard assay conditions with 10 μM ATP at 30°C (top) and kinase activity of both the unphosphorylated and 1-h preautophosphorylated c-Src in the absence or presence of HSP-27 and 5′-AMP for 15 min, using casein as the substrate (bottom). (D) Cos-7 cells were transfected with either the constitutively active form of c-Src, which has phenylalanine substitution at the Tyr-527 site, or an empty vector alone according to the protocol of the manufacturer (Upstate Biotechnology). c-Src was immunoprecipitated with agarose-conjugated v-Src antibody (Ab) and assayed in the presence or absence of HSP-27 and 5′-AMP. (E) Kinase activity of either c-Src (60 kDa) or a fusion protein bearing only the catalytic domain of c-Src (amino acids 279 to 522 in mouse c-Src; 28 kDa) determined in the absence or presence of 5′-AMP, HSP-27, and HSP-32, alone or in combination, as shown in the upper and lower autoradiographs. (F) c-Src autophosphorylation, performed in the absence or presence of 5′-AMP and HSP-27, alone or in combination, analyzed by Western blotting (WB) with either phosphotyrosine antibody or phospho-Tyr-416 c-Src antibody. Unphosphorylated c-Src and HSP-27 phosphorylation in the absence of c-Src (if any) are shown in the first and last lanes, respectively. Positions of the phosphorylated c-Src species with molecular masses of 60, 54, and 52 kDa are numbered 1, 2, and 3, respectively.

FIG. 6

In vivo effect of changing nucleotide concentrations. (A) Isolated adult feline cardiocytes (2 × 10⁵) were cultured for 24 h on laminin-coated 60-mm-diameter culture dishes. Part of the culture plates was used for electroporation in the absence or presence of ATP (9 mM), 5′-AMP (0.5 mM), or both as described in Materials and Methods. A Triton X-100-insoluble fraction was prepared, and the denatured protein samples were used for Western blot analysis using antiphosphotyrosine antibody. Arrows indicate the positions of proteins showing increased tyrosine phosphorylation following the ATP transfer; the solid arrow indicates the position of c-Src in the blot. Positions of molecular mass markers are shown in kilodaltons on the left. (B) Western blot analysis for the same samples was performed with anti-phospho-Tyr-416 c-Src antibody.