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Engagement of β2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils
BBRC Biochemical and Biophysical Research Communications 317 (2004) 1000–1005 www.elsevier.com/locate/ybbrc
Engagement of b2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils Fredrik Melander, Tommy Andersson, and Karim Dib* € Sweden Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmo¨ University Hospital, Entrance 78, SE-205 02 Malmo, Received 23 March 2004
Abstract We found that engagement of b2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the b2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils. Ó 2004 Elsevier Inc. All rights reserved. Keywords: c-Cbl; Human neutrophils; Protein kinase C; Src tyrosine kinase; Ubiquitin ligase
Human polymorphonuclear neutrophils (PMNs) play a crucial role in acute inflammatory reactions [1]. These cells are recruited from the blood-stream to the inflamed tissue by the release of cytokines and chemoattractants which subsequently cause the PMNs to exhibit distinct rolling behavior and adhere to the vessel wall [2]. Firm adhesion of the PMNs is mediated via their b2 integrins, and engagement of those adhesion receptors has also been implicated in the regulation of PMN survival and apoptosis [3]. PMN apoptosis at the site of inflammation is considered to be an essential step in resolution of the inflammatory process [4]. Consequently, detailed information about b2 integrin signaling mechanisms is needed to explain how this adhesion receptor participates in the regulation of PMN extravasation and apoptosis. One of the initial events in b2 integrin-mediated signaling in human PMNs is activation of various tyrosine kinases, such as Src family members, Syk and Pyk2 [5]. These kinases in turn induce tyrosine phosphorylation of numerous cell proteins including the proto-oncogene * Corresponding author. Fax: +46-40-33-73-53. E-mail address: [email protected] (K. Dib).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.147
c-Cbl [5,6]. The multidomain protein c-Cbl has emerged as a key regulator of various tyrosine kinases in hematopoietic cells. The N-terminal portion of c-Cbl can interact directly with a number of tyrosine kinases, among others Syk/ZAP-70 and Src family members [7]. In addition, via its proline-rich domain, c-Cbl can bind adaptor proteins such as Grb2 that contain an SH3 domain. Furthermore, in leukocytes and other types of cells, c-Cbl is phosphorylated on tyrosine residues upon activation of a large number of receptors [7] and can thereby recruit proteins that contain SH2 domains such as the p85 subunit of the phosphatidylinositol 3-kinase. One function of c-Cbl is to catalyze ubiquitination of numerous proteins including the Src family tyrosine kinase Fgr [6], and this can be explained by the findings that c-Cbl contains a RING domain that binds ubiquitin-conjugating enzymes (E2s), and is a E3 ubiquitin ligase [7]. Ubiquitination of proteins depends on both activation of Src tyrosine kinases and tyrosine phosphorylation of c-Cbl [8], and it is a covalent modification that generally triggers rapid degradation of proteins by a proteasomal or a lysosomal pathway [9]. However, it has also been suggested that ubiquitination regulates receptor recycling and the localization of intracellular
F. Melander et al. / Biochemical and Biophysical Research Communications 317 (2004) 1000–1005
proteins [9]. Thus, c-Cbl-induced ubiquitination of intracellular signaling molecules such as Fgr in human PMNs can modify and coordinate receptor-induced intracellular signals. In support of that, the ability of c-Cbl to serve as a multifunctional adaptor protein has been found to have positive affect on intracellular signaling in a number of different cell types [10]. One additional mechanism whereby c-Cbl can possibly modify intracellular signaling is via PKC-mediated serine phosphorylations of the C-terminal end of c-Cbl [11–13]. At present, there is no evidence that b2 integrins mediate serine phosphorylation of c-Cbl in PMNs, although such an event is plausible, since it has been reported that engagement of b2 integrins causes significant activation of PKC in this type of leukocyte [14]. In Jurkat T cells, phorbol ester-induced serine phosphorylation of c-Cbl has been shown to mediate interaction of this protein with adaptor proteins designated 14-3-3 [12], which are dimeric phosphoserine-binding molecules that have been proposed to regulate cell division, signaling, and apoptosis [15]. It was recently observed that transepithelial migration of PMNs results in increased intracellular expression of an anti-apoptotic 143-3 protein and delayed apoptosis [16]. Also, engagement of the glycoprotein GP Ib-IX integrins on platelets has been shown to activate a 14-3-3 protein [17], but nothing is known about a possible b2 integrin-induced activation of this anti-apoptotic protein in human PMNs, even though it has been reported that b2 integrins can modulate the apoptotic response in these leukocytes [3]. To further elucidate how expression of b2 integrins on PMNs is involved in regulation of the inflammatory process, we conducted the present study to determine whether b2 integrins can induce serine phosphorylation of c-Cbl, and whether such modifications are related to distinct intracellular signaling events in PMNs.
Materials and methods Antibodies and chemicals. The mAb 4G10 (mouse anti-phosphotyrosine) was purchased from Upstate Biotechnology (Lake Placid, NY); anti-c-Cbl polyclonal Ab, anti-ubiquitin mAb, and anti-Fgr polyclonal Ab were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fgr mAb was from Wako Bioproducts (Richmond, VA); the anti-phosphoserine mAb (clone 16B4) was from Biomol (SMS, Denmark); and the anti-c-Cbl mAb and the anti-14-3-3 mAb were from BD Biosciences Pharmingen (Stockholm, Sweden). Peroxidase-conjugated Igs were obtained from Dakopatts (Glostrup, Denmark). Protein A–Sepharose, Dextran, and Ficoll–Hypaque were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). PP1, the selective Src family member inhibitor [18], was from Alexis Biochemicals (L€aufelingen, Switzerland). The PKC inhibitor GF-109203X [19] was purchased from Calbiochem (Darmstadt, Germany). Calcein AM was from Molecular Probes Europe (Leiden, The Netherlands). All electrophoresis reagents were obtained from Bio-Rad (Richmond, CA) and [c-32 P]ATP was from Amersham International (Little Chalfont, Bucks, UK). All other chemicals were of analytical grade and were purchased from Sigma–Aldrich (Stockholm, Sweden).
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Isolation of human PMNs. Blood from healthy donors was collected and isolated under endotoxin-free conditions as previously described [6]. The cells were re-suspended in a calcium-containing medium (136 mM NaCl, 4.7 mM KCl, 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 5.0 mM NaHCO3 , 1.1 mM CaCl2 , 0.1 mM EGTA, 5.5 mM glucose, and 20 mM Hepes, pH 7.4) to give a suspension consisting of approximately 97% PMNs. Engagement of b2 integrins. To engage b2 integrins on PMNs, the cells were incubated for 20 min at 37 °C on a surface coated with fibrinogen (a ligand for b2 integrins [20]) in the presence of TNF [21– 23]. Stimulation of PMNs with TNF increases the expression of b2 integrins on the surface of the cells, and it is presumed that these integrins exhibit augmented avidity for their ligands [21]. The reactions were terminated by placing the plates on ice, after which the cells were scraped off and lysed in buffer containing [100 mM Tris–HCl, pH 7.5, 1% NP-40, 5 mM EDTA, 5 mM EGTA, 50 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , and protease inhibitors (20 lg/ml aprotinin, 2.5 mM benzamidine, 2 mM pefabloc, and 1 lg/ml each of pepstatin, leupeptin, and antipain)]. Adhesion assays. The chemical probe calcein AM is non-fluorescent until it is loaded into PMNs where it is cleaved by endogenous esterase to give a highly fluorescent molecule calcein, which has absorption and emission maxima at 495 and 517 nM, respectively. PMNs (5 105 ) were preincubated for 30 min at 37 °C with calcein AM (1 lM) after which the cells were washed, then re-suspended in calcium-containing medium, and then incubated in the presence or absence of PP1 (3 lM) or GF-109203X (10 lM). Thereafter, the PMNs were placed on 96-well plates that have been coated with fibrinogen in the presence of TNF (20 ng/ml). After 20 min, nonadherent cells were removed and the wells were washed with calciumcontaining medium. The percent of adherent cells is obtained by dividing the fluorescence of plates after washing by the fluorescence measured before washing. Immunoprecipitation and Western blotting. Cell lysates were clarified by centrifugation (15,000g, 10 min), and proteins in the supernatants were immunoprecipitated by adding the appropriate antibodies (2–3 lg) for 2 h and thereafter protein A–Sepharose (40 ll of a 50% slurry) for 1 h. The beads were subsequently collected by centrifugation, washed three times in buffer (50 mM Hepes [pH 7.4], 1% NP-40, 150 mM NaCl, and 1 mM Na3 VO4 ), re-suspended in 2 concentrated Laemmli sample buffer, and then boiled under reducing conditions for 5 min. The immunoprecipitated proteins were separated by 8% SDS–PAGE and transferred to polyscreen PVDF transfer membranes. The membranes were blocked in PBS supplemented with 0.2% Tween 20 and 3% BSA and were subsequently incubated for 1 h with a primary antibody (0.5 lg/ml). Thereafter, the membranes were washed three times (15 min each) in PBS supplemented with 0.2% Tween and then incubated for 1 h with peroxidase-conjugated anti-mouse Igs (1:10,000) in PBS supplemented with 0.2% Tween 20 and 3% BSA. The blots were finally washed and antibody binding was visualized by enhanced chemiluminescence. PKC activity in anti-c-Cbl immunoprecipitates. Kinase assays were performed in vitro essentially as reported elsewhere [24]. Briefly, c-Cbl was immunoprecipitated from PMN lysates and the immunoprecipitated proteins were incubated at 25 °C in a kinase assay buffer (20 mM Hepes, pH 7.0, 0.5% Triton X-100, 100 mM MnCl2 , 10 mM MgCl2 , 1 mM DTT, and 0.1 mM Na3 VO4 ) supplemented with a PKC-specific substrate peptide (5 lg) (Calbiochem, Darmstadt, Germany) and 0.25 lCi [c-32 P]ATP in the absence or presence of GF-109203X (10 lM). After 30 min, an aliquot of the supernatant was spotted onto phosphocellulose filter paper which was then washed extensively with kinase assay buffer, and the radioactivity remaining on the filters was measured in a scintillation counter. An aliquot of the supernatant that was free from substrate peptide was also spotted onto phosphocellulose paper and counted to determine the background level of radioactivity.
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Results and discussion In the present study, we found that engagement of b2 integrins on PMNs triggered phosphorylation of the proto-oncogene c-Cbl not only on tyrosine residues [6], but also on serine residues. This was documented by immunoprecipitating c-Cbl from cell lysates of PMNs that had been kept in suspension or allowed to adhere to immobilized fibrinogen in the presence of the pro-inflammatory cytokine TNF, conditions that are known to result in specific ligation of b2 integrins [21–23]. The immunoprecipitates were subsequently analyzed by Western blotting with specific anti-phosphoserine or anti-phosphotyrosine antibodies (Fig. 1A). It has previously been reported that engagement of b2 integrins on PMNs triggers significant activation of PKC [14] and that PKC phosphorylates c-Cbl on serine residues in other types of cells [12], hence we conducted experiments to ascertain whether a member of the PKC family is responsible for the b2 integrin-induced serine phosphorylation of c-Cbl we observed in the current study. This was indeed the case, since we found that PMNs pretreated with the broad range PKC-inhibitor GF109203X (10 lM) exhibited significantly decreased b2 integrin-induced serine phosphorylation of c-Cbl (Fig. 1A). It should be pointed out that the concentration of GF-109203X we used (10 lM) potently blocks classical and novel isoforms of PKC [25] but not the atypical isoforms. Moreover, we noted that GF-109203X had no effect on b2 integrin-induced tyrosine phosphorylation of c-Cbl (Fig. 1A) and, conversely, that inhibition of b2 integrin-induced tyrosine phosphorylation of c-Cbl had no impact on the degree of serine phosphorylation of c-Cbl (Fig. 1A). These results show that there is no interference between the serine phosphorylation and the tyrosine phosphorylation of c-Cbl or vice versa. This is in contrast to studies in which pretreatment of guinea pig PMNs [13] and Jurkat T cells [11,12] with the phorbol ester PMA, a non-physiological component known to activate PKC family members [26], was found to cause serine phosphorylation of c-Cbl that led to impaired receptor-mediated tyrosine phosphorylation of c-Cbl. Similar to our findings, engagement of the Fcc receptor on guinea pig PMNs induced both serine and tyrosine phosphorylation of c-Cbl [13]. As an additional control, we established that neither of the two inhibitors we used (GF-109203X and PP1) affected the ability of PMNs to adhere to fibrinogen (Fig. 1B). Although our present study showed that there was no competition between serine and tyrosine phosphorylation of c-Cbl in PMNs (Fig. 1A), we could not rule out the possibility that serine phosphorylation of c-Cbl influences functions of this protein that are dependent on tyrosine phosphorylation, such as the ability to catalyze mono-ubiquitination of the Fgr tyrosine kinase, which we observed in a previous investigation [6]. However, we
Fig. 1. Engagement of b2 integrins induces both tyrosine and serine phosphorylation of c-Cbl. (A) Suspended PMNs (2 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with PP1 (3 lM), or GF109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Non-pretreated suspended PMNs were used as controls. Thereafter, the PMNs were lysed, and c-Cbl was immunoprecipitated from clarified lysates with an anti-c-Cbl rabbit antiserum. The immunoprecipitated proteins were subjected to 8% SDS–PAGE, then, transferred to a PVDF membrane, and immunoblotted with an anti-phosphoserine mAb (upper panel). The blot was subsequently stripped and re-probed with an anti-phosphotyrosine mAb (middle panel). To confirm that equal amounts of c-Cbl had been immunoprecipitated from each sample, the blot was also re-probed with an anti-c-Cbl mAb (lower panel). Arrows on the left indicate the position of c-Cbl. The blots shown are representative of at least three separate experiments. (B) PMNs (5 105 ) were preincubated for 30 min at 37 °C with the fluorescent probe calcein/AM (1 lM) and then exposed to the indicated inhibitors (PP1, 3 lM; GF-109203X, 10 lM) for 20 min at 37 °C. The cells were subsequently allowed to adhere to a 96-well, fibrinogencoated microplate in the presence of TNF (20 ng/ml). The percent of adherent cells was calculated as described in Materials and methods. The data represent means SD of four separate experiments, each performed in triplicate.
excluded that hypothesis since pretreatment of human PMNs with GF-109203X did not affect the b2 integrininduced mono-ubiquitination of Fgr (Fig. 2, top panel, lane 3) or the phosphorylation/activation of Fgr in adherent PMNs (Fig. 2, middle panel). The observation that serine phosphorylation of c-Cbl did not have an impact on the ubiquitination activity of that protein implies that latter covalent modification plays some other regulatory role. Considering our previous results showing that b2 integrin-induced tyrosine phosphorylation of c-Cbl occurs in parallel with increased c-Cbl-associated tyrosine
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Fig. 2. Inhibition of PKC does not affect the b2 integrin-induced mono-ubiquitination of Fgr. Suspended PMNs (1 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with GF-109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Non-pretreated suspended PMNs were used as controls. Thereafter, the cells were lysed and Fgr was immunoprecipitated from clarified lysates with an antiFgr polyclonal Ab. The immunoprecipitated proteins were resolved by 8% SDS–PAGE, transferred to PVDF membranes, and then immunoblotted with an anti-ubiquitin mAb (upper panel). The blot was then stripped and re-probed, first with an anti-phosphotyrosine mAb (middle panel) and after that with an anti-Fgr mAb (bottom panel). The arrows on the left indicate the positions of mono-ubiquitinated Fgr (75 kDa) and non-ubiquitinated Fgr (58 kDa). The blots shown are representative of three separate experiments.
kinase activity [6], we examined the possibility that the augmented PKC-dependent serine phosphorylation of cCbl is due to elevated anti-c-Cbl-linked activity of PKC. To this end, we measured the extent to which anti-c-Cbl immunoprecipitates obtained from PMNs could phosphorylate a PKC-specific substrate peptide. We detected basal PKC activity in anti-c-Cbl immunoprecipitates from control cells, which agrees with the finding that PKCs can interact with c-Cbl [12]. The notion that a PKC family member causes the kinase activity associated with anti-c-Cbl immunoprecipitates was further confirmed by the observation that the PKC inhibitor GF-109203X blocked PKC activity (Fig. 3). However, we made the somewhat surprising discovery that engagement of b2 integrins did not enhance the c-Cblbound activity of PKC in PMNs, and there are a number of possible explanations for the lack of such an effect in the c-Cbl immunoprecipitates from PMNs with ligated b2 integrins. One reason may be that the association between c-Cbl and PKCs is unstable, as previously suggested [13]. Another, and perhaps more likely alternative, is that the PKC-mediated phosphorylation of serine residues on c-Cbl may induce an association between c-Cbl and a protein that has an inhibitory effect on the c-Cbl-bound PKC activity. A conceivable par-
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Fig. 3. Presence of PKC activity in anti-c-Cbl immunoprecipitates. PMNs (1 107 ) were either kept in suspension (control cells) or incubated for 20 min on fibrinogen-coated plates in the presence of TNF (20 ng/ml). Thereafter, the cells were lysed and c-Cbl was immunoprecipitated from clarified lysates as described in Fig. 1. Western blot analysis shows that similar amounts of c-Cbl had been immunoprecipitated in each case (top panel). Anti-c-Cbl immunoprecipitates were incubated in the presence of [c-32 P]ATP and 5 lg of a PKC-specific substrate peptide in the absence ()) or presence (+) of the PKC inhibitor GF-109203X (10 lM). An in vitro kinase assay was subsequently run for 30 min and an aliquot of the supernatant was spotted onto phosphocellulose paper, which was then washed extensively. The radioactivity remaining on the paper (corresponding to phosphorylation of the substrate peptide) was counted in a scintillation counter and the final value for each sample was calculated by subtracting the background radioactivity bound to the phosphocellulose paper in the absence of peptide substrate. The data are expressed as percent of controls and represent means SD of three separate experiments, each performed in duplicate.
ticipant in such a negative feedback loop is the antiapoptotic protein 14-3-3, since it has been shown that this protein binds to c-Cbl in which the serine-rich motif at the C-terminus has undergone PKC-mediated phosphorylation [11,12]. More importantly, 14-3-3 proteins have also been reported to inhibit the kinase activity of PKCs [27,28]. In addition, an interaction between c-Cbl and a 14-3-3 protein in PMNs is possible since the protein 14-3-3f was recently detected in PMNs [29]. Consequently, to ascertain whether a PKC-dependent association occurs between c-Cbl and a 14-3-3 protein in PMNs, we immunoprecipitated c-Cbl from lysates of PMNs and then analyzed the immunoprecipitates by Western blotting with an anti-14-3-3 antibody. The results revealed that there was a modest association between c-Cbl and 14-3-3 proteins in immunoprecipitates from non-adherent control cells (Fig. 4, top panel) whereas the anti-c-Cbl immunoprecipitates obtained from cells following adhesion-induced engagement of b2 integrins contained a significant amount of 14-3-3 proteins (Fig. 4, top panel). This b2 integrin-induced association between c-Cbl and 14-3-3 proteins is most likely mediated by the PKC-dependent serine phosphorylation
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Fig. 4. b2 Integrin-mediated cell adhesion initiates a PKC-dependent association of 14-4-3 proteins with c-Cbl. Suspended PMNs (1 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with GF109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Thereafter, the cells were lysed and c-Cbl was immunoprecipitated from clarified lysates as described in Fig. 1. The immunoprecipitated proteins were resolved by 8% SDS–PAGE, transferred to PVDF membranes, and then immunoblotted with an anti-14-3-3 mAb (upper panel). The blot was then stripped and re-probed with an anti-c-Cbl mAb (bottom panel). The blot shown is representative of three separate experiments.
of c-Cbl, because it was abolished by pretreatment with GF-109203X, and that inhibitor also blocks the b2 integrin-induced serine phosphorylation of c-Cbl (Fig. 1A). It has been shown that many cell functions, including apoptosis, are affected by the recruitment and activation of 14-3-3 proteins [15,30]. It has been proposed that the role of 14-3-3 proteins in the regulation of apoptosis depends on interactions with other proteins, such as the mitogen-activated protein kinases (MAPK), including p38 MAPK [31,32]. The latter is especially interesting, since it has been suggested that p38 MAPK promotes survival in PMNs by inducing phosphorylation and inactivation of caspases [33]. Accordingly, the present results provide a basis for further investigation of how serine phosphorylation of c-Cbl and the interaction between 14-3-3 protein and c-Cbl are involved in regulation of neutrophil apoptosis.
Acknowledgments We acknowledge the blood donors and thank the staff of the Blood Center of Malm€ o University Hospital for kind and professional help, € and Ms. Patty Odman for linguistic revision of the manuscript. This work was supported by grants from the Swedish Cancer Foundation, the King Gustaf V Memorial Foundation, and the Network for Inflammation Research (to T.A.); the U-MAS Research Foundations, € the Crafoord Foundation, the Osterlund Foundation, the Blood and Defense Network of Lund University (to T.A. and K.D.); the Royal Physiographic Society in Lund, the Kock Foundation, and the Medical Faculty, Lund University (to K.D.).
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Engagement of b2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils Fredrik Melander, Tommy Andersson, and Karim Dib* € Sweden Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmo¨ University Hospital, Entrance 78, SE-205 02 Malmo, Received 23 March 2004
Abstract We found that engagement of b2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the b2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils. Ó 2004 Elsevier Inc. All rights reserved. Keywords: c-Cbl; Human neutrophils; Protein kinase C; Src tyrosine kinase; Ubiquitin ligase
Human polymorphonuclear neutrophils (PMNs) play a crucial role in acute inflammatory reactions [1]. These cells are recruited from the blood-stream to the inflamed tissue by the release of cytokines and chemoattractants which subsequently cause the PMNs to exhibit distinct rolling behavior and adhere to the vessel wall [2]. Firm adhesion of the PMNs is mediated via their b2 integrins, and engagement of those adhesion receptors has also been implicated in the regulation of PMN survival and apoptosis [3]. PMN apoptosis at the site of inflammation is considered to be an essential step in resolution of the inflammatory process [4]. Consequently, detailed information about b2 integrin signaling mechanisms is needed to explain how this adhesion receptor participates in the regulation of PMN extravasation and apoptosis. One of the initial events in b2 integrin-mediated signaling in human PMNs is activation of various tyrosine kinases, such as Src family members, Syk and Pyk2 [5]. These kinases in turn induce tyrosine phosphorylation of numerous cell proteins including the proto-oncogene * Corresponding author. Fax: +46-40-33-73-53. E-mail address: [email protected] (K. Dib).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.147
c-Cbl [5,6]. The multidomain protein c-Cbl has emerged as a key regulator of various tyrosine kinases in hematopoietic cells. The N-terminal portion of c-Cbl can interact directly with a number of tyrosine kinases, among others Syk/ZAP-70 and Src family members [7]. In addition, via its proline-rich domain, c-Cbl can bind adaptor proteins such as Grb2 that contain an SH3 domain. Furthermore, in leukocytes and other types of cells, c-Cbl is phosphorylated on tyrosine residues upon activation of a large number of receptors [7] and can thereby recruit proteins that contain SH2 domains such as the p85 subunit of the phosphatidylinositol 3-kinase. One function of c-Cbl is to catalyze ubiquitination of numerous proteins including the Src family tyrosine kinase Fgr [6], and this can be explained by the findings that c-Cbl contains a RING domain that binds ubiquitin-conjugating enzymes (E2s), and is a E3 ubiquitin ligase [7]. Ubiquitination of proteins depends on both activation of Src tyrosine kinases and tyrosine phosphorylation of c-Cbl [8], and it is a covalent modification that generally triggers rapid degradation of proteins by a proteasomal or a lysosomal pathway [9]. However, it has also been suggested that ubiquitination regulates receptor recycling and the localization of intracellular
F. Melander et al. / Biochemical and Biophysical Research Communications 317 (2004) 1000–1005
proteins [9]. Thus, c-Cbl-induced ubiquitination of intracellular signaling molecules such as Fgr in human PMNs can modify and coordinate receptor-induced intracellular signals. In support of that, the ability of c-Cbl to serve as a multifunctional adaptor protein has been found to have positive affect on intracellular signaling in a number of different cell types [10]. One additional mechanism whereby c-Cbl can possibly modify intracellular signaling is via PKC-mediated serine phosphorylations of the C-terminal end of c-Cbl [11–13]. At present, there is no evidence that b2 integrins mediate serine phosphorylation of c-Cbl in PMNs, although such an event is plausible, since it has been reported that engagement of b2 integrins causes significant activation of PKC in this type of leukocyte [14]. In Jurkat T cells, phorbol ester-induced serine phosphorylation of c-Cbl has been shown to mediate interaction of this protein with adaptor proteins designated 14-3-3 [12], which are dimeric phosphoserine-binding molecules that have been proposed to regulate cell division, signaling, and apoptosis [15]. It was recently observed that transepithelial migration of PMNs results in increased intracellular expression of an anti-apoptotic 143-3 protein and delayed apoptosis [16]. Also, engagement of the glycoprotein GP Ib-IX integrins on platelets has been shown to activate a 14-3-3 protein [17], but nothing is known about a possible b2 integrin-induced activation of this anti-apoptotic protein in human PMNs, even though it has been reported that b2 integrins can modulate the apoptotic response in these leukocytes [3]. To further elucidate how expression of b2 integrins on PMNs is involved in regulation of the inflammatory process, we conducted the present study to determine whether b2 integrins can induce serine phosphorylation of c-Cbl, and whether such modifications are related to distinct intracellular signaling events in PMNs.
Materials and methods Antibodies and chemicals. The mAb 4G10 (mouse anti-phosphotyrosine) was purchased from Upstate Biotechnology (Lake Placid, NY); anti-c-Cbl polyclonal Ab, anti-ubiquitin mAb, and anti-Fgr polyclonal Ab were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fgr mAb was from Wako Bioproducts (Richmond, VA); the anti-phosphoserine mAb (clone 16B4) was from Biomol (SMS, Denmark); and the anti-c-Cbl mAb and the anti-14-3-3 mAb were from BD Biosciences Pharmingen (Stockholm, Sweden). Peroxidase-conjugated Igs were obtained from Dakopatts (Glostrup, Denmark). Protein A–Sepharose, Dextran, and Ficoll–Hypaque were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). PP1, the selective Src family member inhibitor [18], was from Alexis Biochemicals (L€aufelingen, Switzerland). The PKC inhibitor GF-109203X [19] was purchased from Calbiochem (Darmstadt, Germany). Calcein AM was from Molecular Probes Europe (Leiden, The Netherlands). All electrophoresis reagents were obtained from Bio-Rad (Richmond, CA) and [c-32 P]ATP was from Amersham International (Little Chalfont, Bucks, UK). All other chemicals were of analytical grade and were purchased from Sigma–Aldrich (Stockholm, Sweden).
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Isolation of human PMNs. Blood from healthy donors was collected and isolated under endotoxin-free conditions as previously described [6]. The cells were re-suspended in a calcium-containing medium (136 mM NaCl, 4.7 mM KCl, 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 5.0 mM NaHCO3 , 1.1 mM CaCl2 , 0.1 mM EGTA, 5.5 mM glucose, and 20 mM Hepes, pH 7.4) to give a suspension consisting of approximately 97% PMNs. Engagement of b2 integrins. To engage b2 integrins on PMNs, the cells were incubated for 20 min at 37 °C on a surface coated with fibrinogen (a ligand for b2 integrins [20]) in the presence of TNF [21– 23]. Stimulation of PMNs with TNF increases the expression of b2 integrins on the surface of the cells, and it is presumed that these integrins exhibit augmented avidity for their ligands [21]. The reactions were terminated by placing the plates on ice, after which the cells were scraped off and lysed in buffer containing [100 mM Tris–HCl, pH 7.5, 1% NP-40, 5 mM EDTA, 5 mM EGTA, 50 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , and protease inhibitors (20 lg/ml aprotinin, 2.5 mM benzamidine, 2 mM pefabloc, and 1 lg/ml each of pepstatin, leupeptin, and antipain)]. Adhesion assays. The chemical probe calcein AM is non-fluorescent until it is loaded into PMNs where it is cleaved by endogenous esterase to give a highly fluorescent molecule calcein, which has absorption and emission maxima at 495 and 517 nM, respectively. PMNs (5 105 ) were preincubated for 30 min at 37 °C with calcein AM (1 lM) after which the cells were washed, then re-suspended in calcium-containing medium, and then incubated in the presence or absence of PP1 (3 lM) or GF-109203X (10 lM). Thereafter, the PMNs were placed on 96-well plates that have been coated with fibrinogen in the presence of TNF (20 ng/ml). After 20 min, nonadherent cells were removed and the wells were washed with calciumcontaining medium. The percent of adherent cells is obtained by dividing the fluorescence of plates after washing by the fluorescence measured before washing. Immunoprecipitation and Western blotting. Cell lysates were clarified by centrifugation (15,000g, 10 min), and proteins in the supernatants were immunoprecipitated by adding the appropriate antibodies (2–3 lg) for 2 h and thereafter protein A–Sepharose (40 ll of a 50% slurry) for 1 h. The beads were subsequently collected by centrifugation, washed three times in buffer (50 mM Hepes [pH 7.4], 1% NP-40, 150 mM NaCl, and 1 mM Na3 VO4 ), re-suspended in 2 concentrated Laemmli sample buffer, and then boiled under reducing conditions for 5 min. The immunoprecipitated proteins were separated by 8% SDS–PAGE and transferred to polyscreen PVDF transfer membranes. The membranes were blocked in PBS supplemented with 0.2% Tween 20 and 3% BSA and were subsequently incubated for 1 h with a primary antibody (0.5 lg/ml). Thereafter, the membranes were washed three times (15 min each) in PBS supplemented with 0.2% Tween and then incubated for 1 h with peroxidase-conjugated anti-mouse Igs (1:10,000) in PBS supplemented with 0.2% Tween 20 and 3% BSA. The blots were finally washed and antibody binding was visualized by enhanced chemiluminescence. PKC activity in anti-c-Cbl immunoprecipitates. Kinase assays were performed in vitro essentially as reported elsewhere [24]. Briefly, c-Cbl was immunoprecipitated from PMN lysates and the immunoprecipitated proteins were incubated at 25 °C in a kinase assay buffer (20 mM Hepes, pH 7.0, 0.5% Triton X-100, 100 mM MnCl2 , 10 mM MgCl2 , 1 mM DTT, and 0.1 mM Na3 VO4 ) supplemented with a PKC-specific substrate peptide (5 lg) (Calbiochem, Darmstadt, Germany) and 0.25 lCi [c-32 P]ATP in the absence or presence of GF-109203X (10 lM). After 30 min, an aliquot of the supernatant was spotted onto phosphocellulose filter paper which was then washed extensively with kinase assay buffer, and the radioactivity remaining on the filters was measured in a scintillation counter. An aliquot of the supernatant that was free from substrate peptide was also spotted onto phosphocellulose paper and counted to determine the background level of radioactivity.
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Results and discussion In the present study, we found that engagement of b2 integrins on PMNs triggered phosphorylation of the proto-oncogene c-Cbl not only on tyrosine residues [6], but also on serine residues. This was documented by immunoprecipitating c-Cbl from cell lysates of PMNs that had been kept in suspension or allowed to adhere to immobilized fibrinogen in the presence of the pro-inflammatory cytokine TNF, conditions that are known to result in specific ligation of b2 integrins [21–23]. The immunoprecipitates were subsequently analyzed by Western blotting with specific anti-phosphoserine or anti-phosphotyrosine antibodies (Fig. 1A). It has previously been reported that engagement of b2 integrins on PMNs triggers significant activation of PKC [14] and that PKC phosphorylates c-Cbl on serine residues in other types of cells [12], hence we conducted experiments to ascertain whether a member of the PKC family is responsible for the b2 integrin-induced serine phosphorylation of c-Cbl we observed in the current study. This was indeed the case, since we found that PMNs pretreated with the broad range PKC-inhibitor GF109203X (10 lM) exhibited significantly decreased b2 integrin-induced serine phosphorylation of c-Cbl (Fig. 1A). It should be pointed out that the concentration of GF-109203X we used (10 lM) potently blocks classical and novel isoforms of PKC [25] but not the atypical isoforms. Moreover, we noted that GF-109203X had no effect on b2 integrin-induced tyrosine phosphorylation of c-Cbl (Fig. 1A) and, conversely, that inhibition of b2 integrin-induced tyrosine phosphorylation of c-Cbl had no impact on the degree of serine phosphorylation of c-Cbl (Fig. 1A). These results show that there is no interference between the serine phosphorylation and the tyrosine phosphorylation of c-Cbl or vice versa. This is in contrast to studies in which pretreatment of guinea pig PMNs [13] and Jurkat T cells [11,12] with the phorbol ester PMA, a non-physiological component known to activate PKC family members [26], was found to cause serine phosphorylation of c-Cbl that led to impaired receptor-mediated tyrosine phosphorylation of c-Cbl. Similar to our findings, engagement of the Fcc receptor on guinea pig PMNs induced both serine and tyrosine phosphorylation of c-Cbl [13]. As an additional control, we established that neither of the two inhibitors we used (GF-109203X and PP1) affected the ability of PMNs to adhere to fibrinogen (Fig. 1B). Although our present study showed that there was no competition between serine and tyrosine phosphorylation of c-Cbl in PMNs (Fig. 1A), we could not rule out the possibility that serine phosphorylation of c-Cbl influences functions of this protein that are dependent on tyrosine phosphorylation, such as the ability to catalyze mono-ubiquitination of the Fgr tyrosine kinase, which we observed in a previous investigation [6]. However, we
Fig. 1. Engagement of b2 integrins induces both tyrosine and serine phosphorylation of c-Cbl. (A) Suspended PMNs (2 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with PP1 (3 lM), or GF109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Non-pretreated suspended PMNs were used as controls. Thereafter, the PMNs were lysed, and c-Cbl was immunoprecipitated from clarified lysates with an anti-c-Cbl rabbit antiserum. The immunoprecipitated proteins were subjected to 8% SDS–PAGE, then, transferred to a PVDF membrane, and immunoblotted with an anti-phosphoserine mAb (upper panel). The blot was subsequently stripped and re-probed with an anti-phosphotyrosine mAb (middle panel). To confirm that equal amounts of c-Cbl had been immunoprecipitated from each sample, the blot was also re-probed with an anti-c-Cbl mAb (lower panel). Arrows on the left indicate the position of c-Cbl. The blots shown are representative of at least three separate experiments. (B) PMNs (5 105 ) were preincubated for 30 min at 37 °C with the fluorescent probe calcein/AM (1 lM) and then exposed to the indicated inhibitors (PP1, 3 lM; GF-109203X, 10 lM) for 20 min at 37 °C. The cells were subsequently allowed to adhere to a 96-well, fibrinogencoated microplate in the presence of TNF (20 ng/ml). The percent of adherent cells was calculated as described in Materials and methods. The data represent means SD of four separate experiments, each performed in triplicate.
excluded that hypothesis since pretreatment of human PMNs with GF-109203X did not affect the b2 integrininduced mono-ubiquitination of Fgr (Fig. 2, top panel, lane 3) or the phosphorylation/activation of Fgr in adherent PMNs (Fig. 2, middle panel). The observation that serine phosphorylation of c-Cbl did not have an impact on the ubiquitination activity of that protein implies that latter covalent modification plays some other regulatory role. Considering our previous results showing that b2 integrin-induced tyrosine phosphorylation of c-Cbl occurs in parallel with increased c-Cbl-associated tyrosine
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Fig. 2. Inhibition of PKC does not affect the b2 integrin-induced mono-ubiquitination of Fgr. Suspended PMNs (1 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with GF-109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Non-pretreated suspended PMNs were used as controls. Thereafter, the cells were lysed and Fgr was immunoprecipitated from clarified lysates with an antiFgr polyclonal Ab. The immunoprecipitated proteins were resolved by 8% SDS–PAGE, transferred to PVDF membranes, and then immunoblotted with an anti-ubiquitin mAb (upper panel). The blot was then stripped and re-probed, first with an anti-phosphotyrosine mAb (middle panel) and after that with an anti-Fgr mAb (bottom panel). The arrows on the left indicate the positions of mono-ubiquitinated Fgr (75 kDa) and non-ubiquitinated Fgr (58 kDa). The blots shown are representative of three separate experiments.
kinase activity [6], we examined the possibility that the augmented PKC-dependent serine phosphorylation of cCbl is due to elevated anti-c-Cbl-linked activity of PKC. To this end, we measured the extent to which anti-c-Cbl immunoprecipitates obtained from PMNs could phosphorylate a PKC-specific substrate peptide. We detected basal PKC activity in anti-c-Cbl immunoprecipitates from control cells, which agrees with the finding that PKCs can interact with c-Cbl [12]. The notion that a PKC family member causes the kinase activity associated with anti-c-Cbl immunoprecipitates was further confirmed by the observation that the PKC inhibitor GF-109203X blocked PKC activity (Fig. 3). However, we made the somewhat surprising discovery that engagement of b2 integrins did not enhance the c-Cblbound activity of PKC in PMNs, and there are a number of possible explanations for the lack of such an effect in the c-Cbl immunoprecipitates from PMNs with ligated b2 integrins. One reason may be that the association between c-Cbl and PKCs is unstable, as previously suggested [13]. Another, and perhaps more likely alternative, is that the PKC-mediated phosphorylation of serine residues on c-Cbl may induce an association between c-Cbl and a protein that has an inhibitory effect on the c-Cbl-bound PKC activity. A conceivable par-
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Fig. 3. Presence of PKC activity in anti-c-Cbl immunoprecipitates. PMNs (1 107 ) were either kept in suspension (control cells) or incubated for 20 min on fibrinogen-coated plates in the presence of TNF (20 ng/ml). Thereafter, the cells were lysed and c-Cbl was immunoprecipitated from clarified lysates as described in Fig. 1. Western blot analysis shows that similar amounts of c-Cbl had been immunoprecipitated in each case (top panel). Anti-c-Cbl immunoprecipitates were incubated in the presence of [c-32 P]ATP and 5 lg of a PKC-specific substrate peptide in the absence ()) or presence (+) of the PKC inhibitor GF-109203X (10 lM). An in vitro kinase assay was subsequently run for 30 min and an aliquot of the supernatant was spotted onto phosphocellulose paper, which was then washed extensively. The radioactivity remaining on the paper (corresponding to phosphorylation of the substrate peptide) was counted in a scintillation counter and the final value for each sample was calculated by subtracting the background radioactivity bound to the phosphocellulose paper in the absence of peptide substrate. The data are expressed as percent of controls and represent means SD of three separate experiments, each performed in duplicate.
ticipant in such a negative feedback loop is the antiapoptotic protein 14-3-3, since it has been shown that this protein binds to c-Cbl in which the serine-rich motif at the C-terminus has undergone PKC-mediated phosphorylation [11,12]. More importantly, 14-3-3 proteins have also been reported to inhibit the kinase activity of PKCs [27,28]. In addition, an interaction between c-Cbl and a 14-3-3 protein in PMNs is possible since the protein 14-3-3f was recently detected in PMNs [29]. Consequently, to ascertain whether a PKC-dependent association occurs between c-Cbl and a 14-3-3 protein in PMNs, we immunoprecipitated c-Cbl from lysates of PMNs and then analyzed the immunoprecipitates by Western blotting with an anti-14-3-3 antibody. The results revealed that there was a modest association between c-Cbl and 14-3-3 proteins in immunoprecipitates from non-adherent control cells (Fig. 4, top panel) whereas the anti-c-Cbl immunoprecipitates obtained from cells following adhesion-induced engagement of b2 integrins contained a significant amount of 14-3-3 proteins (Fig. 4, top panel). This b2 integrin-induced association between c-Cbl and 14-3-3 proteins is most likely mediated by the PKC-dependent serine phosphorylation
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Fig. 4. b2 Integrin-mediated cell adhesion initiates a PKC-dependent association of 14-4-3 proteins with c-Cbl. Suspended PMNs (1 107 ) were (+) or were not ()) pretreated for 20 min at 37 °C with GF109203X (10 lM) after which the cells were allowed to adhere to a fibrinogen-coated surface in the presence of TNF (20 ng/ml) for 20 min. Thereafter, the cells were lysed and c-Cbl was immunoprecipitated from clarified lysates as described in Fig. 1. The immunoprecipitated proteins were resolved by 8% SDS–PAGE, transferred to PVDF membranes, and then immunoblotted with an anti-14-3-3 mAb (upper panel). The blot was then stripped and re-probed with an anti-c-Cbl mAb (bottom panel). The blot shown is representative of three separate experiments.
of c-Cbl, because it was abolished by pretreatment with GF-109203X, and that inhibitor also blocks the b2 integrin-induced serine phosphorylation of c-Cbl (Fig. 1A). It has been shown that many cell functions, including apoptosis, are affected by the recruitment and activation of 14-3-3 proteins [15,30]. It has been proposed that the role of 14-3-3 proteins in the regulation of apoptosis depends on interactions with other proteins, such as the mitogen-activated protein kinases (MAPK), including p38 MAPK [31,32]. The latter is especially interesting, since it has been suggested that p38 MAPK promotes survival in PMNs by inducing phosphorylation and inactivation of caspases [33]. Accordingly, the present results provide a basis for further investigation of how serine phosphorylation of c-Cbl and the interaction between 14-3-3 protein and c-Cbl are involved in regulation of neutrophil apoptosis.
Acknowledgments We acknowledge the blood donors and thank the staff of the Blood Center of Malm€ o University Hospital for kind and professional help, € and Ms. Patty Odman for linguistic revision of the manuscript. This work was supported by grants from the Swedish Cancer Foundation, the King Gustaf V Memorial Foundation, and the Network for Inflammation Research (to T.A.); the U-MAS Research Foundations, € the Crafoord Foundation, the Osterlund Foundation, the Blood and Defense Network of Lund University (to T.A. and K.D.); the Royal Physiographic Society in Lund, the Kock Foundation, and the Medical Faculty, Lund University (to K.D.).
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