Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1)

Article metrics

Abstract

Activation of c-Jun N-terminal kinase (JNK) by Fas ligation is caspase-dependent, suggesting that caspases may regulate activators of the JNK pathway. Here, we report that an upstream activator of JNK, hematopoietic progenitor kinase 1 (HPK1), was cleaved during apoptosis. Cleavage of HPK1 was blocked by peptide inhibitors for caspases. HPK1 was efficiently processed by recombinant caspase 3 in vitro. A conserved caspase recognition site, DDVD (amino acids 382 – 385), was found in the HPK1 protein sequence. By testing HPK1 proteins with in vivo and in vitro cleavage assays, we showed that aspartic acid residue 385 is the target for caspases. HPK1 cleavage separated the amino N-terminal kinase domain from the carboxyl C-terminal regulatory domain, and enhanced HPK1 kinase activity. Unlike the full-length HPK1, the N-terminal cleaved product failed to bind adaptor molecules Grb2 (growth factor receptor-bound protein 2) and Crk (CT10 regulator of kinase). The C-terminal fragment, although having three proline-rich domains, bound to Grb2 and Crk less efficiently than the full-length HPK1 protein. Taken together, the cleavage of HPK1 by caspase profoundly changed its biochemical properties.

Introduction

Apoptosis is important in regulating development and maintaining homeostasis in multicellular organisms (reviewed in Steller, 1995; Thompson, 1995). Apoptosis is positively and negatively regulated by genetic and biochemical programs (reviewed in Steller, 1995). In recent years, the molecular mechanisms of apoptosis have gradually been unfolded. Caspases, aspartate-directed cysteine proteases, are required for apoptosis. The blockage of caspase activation by peptide inhibitors or by viral proteins, such as the pox virus protein CrmA or baculovirus p35, suppresses apoptosis progression (reviewed in Salvesen and Dixit, 1997). Cleavage by caspases may enhance the biochemical activity of their substrates, e.g., caspases themselves (Salvesen and Dixit, 1997) and protein kinase C δ (Emoto et al., 1995). Cleavage by caspases can also diminish normal functions of the substrates, such as poly-(ADP-ribose) polymerase (PARP) (Lazebnik et al., 1994), DNA-dependent protein kinase (Song et al., 1996), MDM2 (Erhardt et al., 1997), p21Cip1/Waf1, and p27Kip1 (Levkau et al., 1998). In addition, cleavage of nuclear lamin (Lazebnik et al., 1995), gelsolin (Kothakota et al., 1997), and focal adhesion kinase (FAK) (Wen et al., 1997) by caspases is involved in the morphological changes found in apoptotic cells.

The c-Jun N-terminal kinase (JNK) family, also called stress-activated protein kinase (SAPK), belongs to the mitogen-activated protein kinase (MAPK) superfamily (reviewed in Ip and Davis, 1998). JNK is activated in apoptosis induced by various stimuli (reviewed in Ip and Davis, 1998). Activation of the JNK pathway can lead to cell death (Xia et al., 1995; Chen et al., 1996b; Ichijo et al., 1997). Interference with the JNK pathway by dominant-negative kinases or an antisense to JNK suppresses apoptosis (Xia et al., 1995; Chen et al., 1996b, 1998; Verheij et al., 1996; Zanke et al., 1996; Goillot et al., 1997; Seimiya et al., 1997; Kasibhatla et al., 1998). However, reports on the importance of JNK in Fas-mediated apoptosis were controversial (Brenner et al., 1997; Goillot et al., 1997; Lenczowski et al., 1997). The exact mechanism by which JNK was activated by apoptotic stimuli was still unclear. Previously, we and others have shown that JNK can be activated by oxidative stresses induced by different apoptotic stimuli, and that this JNK activation is independent of caspase activation (Park et al., 1996; Chen et al., 1998). Nevertheless, in Fas-mediated apoptosis, JNK induction is suppressed by a caspase inhibitor (Chen et al., 1996b; Lenczowski et al., 1997), indicating that certain JNK activator(s) may be regulated by caspases.

Like other MAP kinases, JNK is regulated by a kinase cascade, MAPK kinase kinase (MAP3K/MEKK)→MAPK kinase (MAP2K/MKK)→MAPK (JNK) (Kyriakis and Avruch, 1996). To date, two MAP2K/MKKs, MKK4/SEK1 and MKK7, are known to activate JNK preferentially (Tournier et al., 1997; Yao et al., 1997b). Multiple MAP3K/MEKKs were found to activate the JNK pathway via MKK4/SEK1 or MKK7. These MEKK-like kinases include MEKK1 – 4, MAPKKK5/ASK1, TAK1, Tpl-2/Cot, MLK3/SPRK, and MUK/DLK (reviewed in Fanger et al., 1997). In addition, several yeast STE20-like kinases, such as p21-GTPase (Rac1/Cdc42)-activated kinases (PAKs) and germinal center kinase (GCK), were also shown to activate the JNK pathway (Bagrodia et al., 1995; Pombo et al., 1995). We and others have cloned and characterized the hematopoietic progenitor kinase 1 (HPK1) (Hu et al., 1996; Kiefer et al., 1996), which is homologous to GCK. Our results indicate that HPK1 regulates the JNK pathway through the HPK1□thinsp;? MEKK1, TAK1 → MKK4/SEK, MKK7□thinsp;? JNK cascade (Hu et al., 1996; Wang et al., 1997; Zhou et al., 1999). HPK1 may mediate extracellular signals through interaction with Src-homology 3 (SH3) domain-containing adaptor molecules (Anafi et al., 1997; Oehrl et al., 1998; Ling et al., 1999). Here we report that HPK1 was cleaved by caspase activity during apoptosis. This cleavage separated HPK1's kinase domain from its C-terminal regulatory domain, and enhanced its kinase activity. Cleavage of HPK1 by caspases also greatly reduced its ability to associate with adaptor molecules Grb2 and Crk.

Results

Caspase-mediated cleavage of HPK1 during Fas ligation-induced apoptosis

Our previous finding shows that both γ radiation and anti-Fas treatment induce persistent JNK activation in Jurkat cells, but the mechanisms of JNK induction in these two events are different. Fas-mediated JNK activation was suppressed by a caspase inhibitor, z-VAD-FK. In contrast, γ radiation-induced JNK activation was not suppressed by z-VAD-FK (Figure 1). The suppression of Fas-mediated JNK activation by a caspase inhibitor suggests that the JNK pathway can be activated by a caspase-dependent mechanism.

Figure 1
figure1

Fas-mediated, but not γ radiation-induced, JNK activation is caspase dependent. Jurkat T cells were preincubated with z-VAD-FK (100 μM) for 2 h, and then treated with either γ radiation (100 Gy) or Fas ligation (CH-11, 100 ng/ml) for 3 h. The cells were harvested and endogenous JNK activity was examined by immunocomplex kinase assays using an anti-JNK1 antibody. The results of the kinase assays were quantitated by a densitimeter, data presented are the means and standard deviations of three measurements

HPK1 is a JNK upstream activator, which regulates JNK through the HPK1→MEKK1, TAK1→MKK4, MKK7→JNK pathway (Hu et al., 1996; Wang et al., 1997). By examining the protein sequence of human HPK1, we located one consensus caspase target site between the first and second proline-rich motifs (Figure 2a). This sequence, DDVD (amino acids 382 – 385), is homologous to the substrate sequence DEXD (X stands for any amino acid) for a group of caspases, including caspases 2, 3, and 7 (Salvesen and Dixit, 1997). Processing of HPK1 at this site would separate the N-terminal kinase domain from the C-terminal domain, with predicted molecular weights of 43 kD and 54 kD, respectively (Figure 2a).

Figure 2
figure2

Caspase-mediated HPK1 cleavage during Fas ligation-induced apoptosis. (a) A consensus recognition site (underlined) for caspases was found in the human HPK1 protein sequence. The triangle indicates the putative caspase cleavage site. The regions recognized by three different anti-HPK1 antibodies (Ab484, Ab2025, and Ab1404) are shown. Black bars represent proline-rich motifs. (b) Jurkat cells were treated with anti-Fas (CH-11, 100 ng/ml), and were collected at different time points as indicated. Endogenous HPK1 levels were analysed by SDS – PAGE and Western blotting using an anti-HPK1 antibody (Ab2025). (c) Jurkat cells were treated with anti-Fas in the presence or absence of the caspase inhibitors z-VAD-FK (100 μM) or z-DEVD-FK (50 μM). Endogenous HPK1 proteins were analysed by SDS – PAGE and Western blotting using an anti-N-terminus antibody (Ab2025) or an anti-C-terminus antibody (Ab1404)

To examine the possible processing of HPK1 during apoptosis, Jurkat human T cells were treated with an anti-Fas antibody (CH-11), and endogenous HPK1 in treated cells was examined by Western blotting using an anti-N-terminal HPK1 antibody (Figure 2a,b). We found that a new protein band with an apparent molecular weight of 43 kD appeared on the Western blot (Figure 2b), suggesting HPK1 is cleaved during apoptosis. To verify if the HPK1 cleavage was mediated by caspases, Jurkat T cells were treated with the anti-Fas antibody in the presence or absence of a pan-caspase inhibitor, z-VAD-FK or a selective caspase inhibitor, z-DEVD-FK. The treated cell lysates were then analysed by Western blotting with antibodies against either the N-terminus (Ab2025) or the C-terminus (Ab1404) of HPK1 (Figure 2c). Both anti-HPK1 antibodies recognized one new protein band in anti-Fas-treated cells, which was absent in untreated cells. These protein bands had molecular weights corresponding to the predicted cleavage products (Figure 2a,c), suggesting that HPK1 was processed at the putative cleavage site. Furthermore, the HPK1 cleavage was blocked by co-treatment of the cells with either caspase inhibitor (Figure 2c), suggesting that the cleavage was mediated through a caspase-dependent pathway.

Aspartic acid residue 385 is the caspase cleavage site in HPK1

Sequence examination and the Western blot analysis of the cleaved HPK1 proteins suggested that DDVD385 is a possible caspase recognition site in HPK1. Inhibition of HPK1 cleavage by the caspase inhibitor, z-DEVD-FK, also suggests that HPK1 cleavage is dependent on a DEVD-oriented caspase (or caspases) such as caspase 2, 3 and/or 7. To verify that HPK1 is a direct target for caspases, the in vitro translated 35S-methionine-labeled HPK1 protein was used as a substrate for caspase assays. Recombinant caspase 3, which has a DEVD-oriented substrate specificity (Salvesen and Dixit, 1997), was used in the assays. Caspase 3 processed HPK1 efficiently, since the full-length HPK1 proteins were almost completely cleaved in 30 min (Figure 3a). This result indicates that HPK1 is a direct substrate for caspase 3, and suggests that HPK1 may be cleaved by caspase-3 like proteases in vivo.

Figure 3
figure3

Aspartic acid residue 385 is the caspase cleavage site in HPK1 (a) in vitro translated 35S-labeled Flag-tagged HPK1 proteins were immunoprecipitated from reticulocyte lysate using an anti-Flag antibody (M2), and incubated with recombinant caspase 3 for indicated times. Reaction mixtures were resolved by SDS – PAGE and analysed by autoradiography. (b) HeLa cells were transfected with various HPK1 constructs for 8 h. Transfected cells were incubated in complete medium for 12 – 14 h, then treated with UV-C (30 J/m2). Irradiated cells were then incubated with or without an anti-Fas antibody (CH-11, 100 ng/ml) for 4 h. Expression of HPK1 proteins was examined by Western blotting using an anti-HPK1 antibody (Ab484). (c) in vitro translated 35S-labeled Flag-tagged HPK1 proteins were immunoprecipitated from reticulocyte lysate using an anti-Flag antibody (M2), and incubated with caspase 3 for 3 h in the presence or absence of z-DEVD-FK (50 nM). Reaction mixtures were resolved by SDS – PAGE and analysed by autoradiography. (d) Left panel: 293 cells were transfected with empty vector, HPK1, or HPK1-(D385) expressing plasmid (1 μg each). Right panel: 293 cells were transfected with different combinations of expressing plasmids including empty vector plus HPK1 (1 μg each), HPK1 plus HA-JNK1 (1 μg each), or HPK1-(D385N) plus HA-JNK1 (1 μg each). HPK1 and HA-JNK1 activities in the transfected cells were determined by immunocomplex assays as described in Materials and methods

Our data suggest that the DDVD385 motif in HPK1 is a potential target for caspases. Also, we found an identical DDVD motif (amino acids 381 – 384) in murine HPK1 protein sequence (Kiefer et al., 1996), suggesting that this motif may be a conserved caspase cleavage site. To examine whether DDVD385 is the real caspase recognition site, we generated an HPK1 mutant by changing the critical aspartic residue (D385) to an asparagine (N). The susceptibility of the HPK1-(D385N) protein to caspases was tested by an in vivo system. HeLa cells were transfected with different HPK1 constructs. The transfected cells were irradiated with a low dose of UV-C (30 J/m2) to sensitize them to Fas ligation (Rehemtulla et al., 1997), and were then incubated with or without an anti-Fas antibody. The expression and processing of the transfected HPK1 were examined by Western blot analyses using an anti-HPK1 (N-terminus) antibody (Figure 3b). Wild-type HPK1 was not expressed well in HeLa cells, but the kinase-dead HPK1-M(46) was expressed efficiently, and the level of the N-terminal cleaved fragment (HPK1-N) increased after Fas ligation (Figure 3b), indicating that HPK1-M(46) was processed by caspases. The N-terminal cleaved fragment was not detected in cells transfected with HPK1-(D385N) after Fas ligation, suggesting that the mutated protein was resistant to caspase cleavage. This result also suggests that the sequence, DDVD385, is the major target of caspases in a cellular context.

In vitro transcribed, translated, and 35S-methionine-labeled HPK1 proteins were also tested with an in vitro cleavage assay using recombinant caspase 3 proteins. The wild-type HPK1 protein was cleaved by caspase 3, and the cleavage was inhibited by z-DEVD-FK, indicating that the cleavage is mainly caused by caspase 3, but not by contaminated proteases from reticulocyte lysate. In contrast, HPK1-(D385N) was completely resistant to caspase 3, even after a 3 h incubation (Figure 3c). This result is consistent with the in vivo experiments (Figure 3b), suggesting that aspartic acid 385 is the target for caspases in the HPK1 protein. The HPK1-(D385N) mutant showed similar kinase activity and JNK-activating ability as the wild-type HPK1 (Figure 3d), indicating that the point mutation only affected HPK1's susceptibility to caspases but had no other global effect on HPK1.

A potential reciprocal regulation between HPK1 and caspases

Because the HPK1 cleavage separated the kinase domain from the C-terminal regulatory domain, we examined whether the cleavage of HPK1 affects its kinase activity. HPK1 was immunoprecipitated from untreated or anti-Fas-treated Jurkat cells by an antibody (Ab484) that recognized the N-terminus of HPK1 proteins (Figure 4a), and then examined by in vitro kinase assays using myelin basic protein (MBP) as a substrate. HPK1 activity increased in anti-Fas treated cells when compared with untreated cells (Figure 4a). This activation was suppressed in the presence of the caspase inhibitor z-VAD-FK during Fas ligation, suggesting that the cleavage of HPK1 by caspases enhances its kinase activity.

Figure 4
figure4

A potential reciprocal regulation between HPK1 and caspases. (a) Jurkat cells were treated with anti-Fas (CH-11, 100 ng/ml) in the presence or absence of z-VAD-FK (100 μM). Endogenous HPK1 proteins were analysed by SDS – PAGE and Western blotting using an anti-N-terminal HPK1 antibody (Ab484). HPK1 activity was isolated with Ab484 and examined by immunocomplex kinase assays using MBP as a substrate. (b) HeLa cells were transfected with a plasmid encoding HPK1 or HPK1-M(46). Transfected cells were then incubated in complete medium with or without z-VAD-FK (100 μM) for 24 h. Cells were collected and the expression of HPK1 proteins was examined by Western blotting using an anti-HPK1 antibody (Ab484)

It is interesting to note that the expression level of wild-type HPK1 was much less than that of kinase-dead HPK1-M(46) in transfected HeLa cells (Figure 3b). One possibility is that overexpression of HPK1 may activate the JNK pathway and downstream apoptotic signaling, subsequently, induce apoptosis in HeLa cells. Therefore, wild-type HPK1 may either induce its own cleavage or stop its own expression by inducing caspase activation and apoptosis. To test this possibility, we expressed both HPK1 and HPK1-M(46) in HeLa cells in the presence or absence of the caspase inhibitor, z-VAD-FK. The addition of the caspase inhibitor greatly increased the expression level of full-length HPK1, while only marginally increased the HPK1-M(46) levels (Figure 4b). This result suggests that HPK1's kinase activity induced caspase activation and its own degradation.

The N-terminal HPK1 fragment retains JNK activating ability

Since HPK1 is an upstream JNK activator and the JNK pathway is involved in apoptotic signaling, we then examined whether cleavage of HPK1 affects its ability to induce JNK activation. Co-expression of HPK and JNK enhanced JNK kinase activity (Figure 5). HPK1-N(1-385), corresponding to the N-terminal HPK1 cleaved product, induced stronger JNK activation than the full-length HPK1 did (Figure 5). The kinase-dead HPK1-M(46) and C-terminal fragment HPK1-C(386-833) failed to activate JNK (Figure 5). This result suggests that cleavage of HPK1 may enhance its JNK activating ability.

Figure 5
figure5

N-terminal HPK1 fragment retains JNK activating ability. 293 cells were transfected with HA-JNK1 (1 μg) in combination with indicated plasmids (1 μg each). The total amount of DNA was normalized with empty vectors. Thirty-six hours after removing the transfection mixture, cell lysates were prepared and JNK activity was immunoprecipitated by an anti-HA antibody (12CA5) and examined by in vitro kinase assays. Expression of HA-JNK was examined by Western blotting using an anti-HA antibody

Cleavage of HPK1 reduces its interaction with adaptor molecules

We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via proline-rich motifs in the C-terminal regulatory region (Anafi et al., 1997; Oehrl et al., 1998; Ling et al., 1999). Cleavage of HPK1 by caspases separates the N-terminus of HPK1 (containing kinase domain and the first proline-rich motif) from the C-terminus (containing the remaining three proline-rich motifs (Figure 2a). Therefore, the cleavage of HPK1 may affect its ability to associate with adaptor molecules and, very likely, change HPK1 localization and functions. We tested whether cleaved HPK1 fragments bind to adaptor molecules Grb2 and Crk. Cell lysates isolated from Jurkat cells treated with or without anti-Fas were incubated with GST-Crk (or GST-Grb2) fusion proteins, and then were affinity-purified by GSH-sepharose beads followed by repeated washes. The precipitated complexes were subjected to SDS – PAGE and Western blot analyses using anti-HPK1 antibodies (anti-N-terminus Ab484 and anti-C-terminus Ab1404). The full-length HPK1 in either untreated or anti-Fas treated cell lysate bound to GST-Crk and GST-Grb2 proteins. The N-terminal cleaved fragment failed to bind GST-Crk and GST-Grb2 (Figure 6a). The C-terminal cleaved fragment of HPK1 retained the Crk (or Grb2)-binding property (Figure 6b); however, its binding to Crk (or Grb2) was much less efficient than the full-length HPK1 protein (Figure 6b).

Figure 6
figure6

Decreases in HPK1-adaptor molecules interaction after caspase cleavage. (a and b) Cell lysates (500 μg) isolated from Jurkat cells treated with or without anti-Fas (CH-11, 100 ng/ml; 4 h) were incubated with GST-Grb2 (or GST-Crk) fusion proteins (20 μg). The protein complexes were affinity-purified by GSH-sepharose beads followed by repeated washes as described in Materials and methods. The precipitated complexes were subjected to SDS – PAGE and Western blot analyses using anti-HPK1 antibodies (a, anti-N-terminus Ab484; b, anti-C-terminus Ab1404). Crude cell lysate (50 μg) from untreated and anti-Fas treated cells were used as controls

Discussion

Both caspases and the JNK pathway participate in apoptotic signaling induced by various stimuli. However, the relative order of JNK and caspases in apoptotic signaling differ in apoptosis induced by distinct stimuli. In apoptosis induced by growth factor withdrawal, radiation, or anti-cancer drugs, JNK activation occurs in the absence of caspase activity (Chen et al., 1996b, 1998; Park et al., 1996). In contrast, caspase activity is required for JNK activation in Fas-mediated apoptosis (Chen et al., 1996b; Lenczowski et al., 1997). The importance of the JNK pathway in Fas-mediated apoptosis is controversial (Brenner et al., 1997; Goillot et al., 1997; Lenczowski et al., 1997).

Recently, several JNK upstream activators including MEKK1, PAK2/hPAK65, and Mst1 were shown to be substrates of caspases (Cardone et al., 1997; Rudel and Bokoch, 1997; Deak et al., 1998; Graves et al., 1998; Widmann et al., 1998). The similarities among these reports are that the cleavage separates the kinase domain from the regulatory domain, and enhances the kinase activity (Cardone et al., 1997; Rudel and Bokoch, 1997; Deak et al., 1998; Graves et al., 1998; Widmann et al., 1998). The cleaved kinases have either the same or enhanced ability to activate JNK. In this report, we show that HPK1 was also cleaved by caspases in Fas-mediated apoptosis. The cleavage of HPK1 separated the kinase domain from the C-terminal regulatory domain, enhanced its kinase activity, and may enhance downstream JNK activation. We were unable to block Fas-mediated JNK activation by kinase-dead HPK1 mutants (data not shown). One possibility is that in the presence of cleavage and activation of multiple JNK upstream kinases by caspases, blocking of HPK1 alone is not sufficient to suppress JNK activation. In addition to HPK1, we found that other two HPK1-like, JNK activating kinases, GCK-like kinase (GLK) and HPK1/GCK homologous kinase (HGK) (Diener et al., 1997; Yao et al., 1997a), were also cleaved by caspase activity during Fas-mediated apoptosis (data not shown). Taken together, our and others' data suggest that the cleavage of JNK upstream regulators is a conserved mechanism, which may mediate JNK activation and other downstream effects. It has been shown that JNK activation is required for caspase activation by certain stimuli (Seimiya et al., 1997; Shiah et al., 1998). It is possible that apoptotic signaling is a circuit that death stimuli can enter at either the JNK pathway or the caspase cascade, and the signaling circuit may amplify the death signal through the reciprocal interaction between these two signaling modules.

How the cleavage of HPK1 enhances its kinase activity is intriguing. The regulatory domain of S. pombe PAK1 has been shown to inhibit its own kinase activity, and the binding of Cdc42 GTPase releases the inhibitory effect. We found that the HPK1 kinase domain construct is more active than the full-length protein, suggesting that the C-terminus of HPK1 may also serve as an inhibitory domain. Cleavage of HPK1 may relieve this self-inhibitory effect. One interesting structural feature of HPK1 is its four proline-rich motifs (putative SH3 domain-binding sites) in its regulatory region (Hu et al., 1996). We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via these proline-rich motifs (Anafi et al., 1997; Oehrl et al., 1998; Ling et al., 1999). Co-expression of Crk with CrkL enhances HPK1 activity through mechanisms yet unknown (Ling et al., 1999). It is possible that binding of adaptors to the proline-rich motifs of HPK1 also relieves the self-inhibitory effect.

Cleavage of HPK1 by caspases separated the N-terminus of HPK1 (containing the first proline-rich domain) from the C-terminus (containing proline-rich domains 2, 3 and 4), and decreased its binding to adaptor molecules Grb2 and Crk. It has been shown that interaction between HPK1 and adaptor molecules may play a role in recruiting HPK1 to the surface receptor complexes (Anafi et al., 1997; Ling et al., 1999). The cleavage of HPK1 greatly reduced its ability to associate with Grb2 and Crk and, very likely, may change HPK1 localization and functions. Previously, we also observed that HPK1 mutants interfere with IL-2 promoter activation induced by T-cell activation signals (Ling et al., 1999). This result suggests that HPK1 may mediate T-cell receptor signaling. Our data indicate that cleaved HPK1 is still capable of activating the JNK pathway; however, whether and how the cleavage will affect HPK1's other downstream functions remains uncertain. It will be important to examine whether cleavage of HPK1 disables other T-cell activation signaling but specifically leaves the JNK pathway in activated condition. If that is the case, it will be a novel mechanism that caspases inhibit mitogenic signaling and, at the same time, leave the apoptotic signaling intact.

Materials and methods

Cells, antibodies, and reagents

Human Jurkat T cells (clone J.LEI) were cultured as described (Chen et al., 1996a). HeLa cells and human embryonic kidney 293 cells were cultured in DMEM supplemented with 10% fetal calf serum and streptomycin/penicillin. Rabbit anti-JNK1 antibody (Ab101) and anti-HPK1 antibodies (Ab2025, Ab484, and Ab1404) were described previously (Chen et al., 1996b; Hu et al., 1996; Ling et al., 1999). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody was obtained from Sigma. The anti-Flag (M2) and anti-HA (12CA5) antibodies were purchased from Kodak and Boehringer Mannheim, respectively. Protein A and G-agarose beads were purchased from Bio-Rad and Santa Cruz, respectively. Caspase inhibitors, z-VAD-FK and z-DEVD-FK, and the anti-Fas antibody (CH-11) were purchased from Kamiya Biomedical. Myelin basic protein (MBP) was purchased from Gibco. Caspase 3 was purchased from Pharmingen.

Plasmids

Plasmids of GST – c-Jun(1 – 79), HA – JNK1, MEKK1, Flag – HPK1, Flag – HPK1-M(46), were described previously (Hu et al., 1996; Meyer et al., 1996). Flag – HPK1(D385N) was constructed by changing HPK1's aspartic acid residue 385 to an asparagine using polymerase chain reaction (PCR)-mediated site-directed mutagenesis, and inserted into the pCR3.1 expression plasmid (Invitrogen). Flag – HPK1-N(1-385) and Flag – HPK1-C(386-833) were generated by PCR and inserted into the pTOPO3.1 vector (Invitrogen). The bacteria-expressing constructs of fusion proteins GST – Crk and GST – Grb2 were kindly provided by SM Feller (Bavarian Julius-Maximilians University, Wuzburg, Germany) and by EY Skolnik (New York University Medical Center, New York, USA), respectively.

In vitro caspase cleavage assays

35S-methionine-labeled Flag-HPK1 proteins (wild-type and the D385N mutant) were synthesized by an in vitro transcription and translation kit (Promega Biotech) as described previously (Ling et al., 1999). Labeled HPK1 proteins were immunoprecipitated from the reaction mixture with an anti-Flag antibody (M2) and protein G-agarose beads to remove endogenous proteases in reticulocyte lysate. The precipitated proteins were incubated in 50 μl of caspase reaction buffer (10 mM HEPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, pH 7.2) with recombinant caspase 3 (100 ng). The reaction was carried out at 37°C for indicated times, and terminated by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS – PAGE (10%) and analysed by autoradiography.

Cell transfection and cell extracts preparation

The cells were plated 24 h before transfection at a density of 1.5×105 per 35-mm well. The 293 cells were transfected by a calcium phosphate precipitation protocol (Specialty Media), and HeLa cells were transfected using the Lipofectamine Plus reagent (Gibco) according to the manufacturer's instructions.

Whole cell lysate was prepared by suspending 2×106 cells in 150 μl lysis buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM EGTA, 50 mM glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM DTT, 2 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1 mM Na3VO4). The cell lysates were kept on ice and vigorously vortexed every 5 min for 20 min. The lysate was cleared by centrifugation at 15 000 g for 3 min, and stored at −80°C.

Immunocomplex kinase assays

The immunocomplex kinase assay for JNK activity was carried out as described (Chen et al., 1996b). For the HPK1 assays, endogenous HPK1 was precipitated with a specific antibody and protein A-agarose beads (Bio-Rad) in lysis buffer at 4°C for 2 h. The precipitates were washed twice with lysis buffer, twice with LiCl buffer (500 mM LiCl, 100 mM Tris-Cl [pH 7.6], and 0.1% Triton X-100), and twice with kinase buffer (20 mM 4-morpholinepropane-sulfonic acid [MOPS; pH 7.6], 2 mM EGTA, 10 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothrietol [DTT], and 1 mM Na3VO4), then mixed in 30 μl of kinase buffer containing 50 μM of ATP, and 10 μCi of [γ-32P]ATP. Five μg of MBP was added per reaction as a substrate. The reaction was performed at 30°C for 30 min, then terminated by adding SDS sampling buffer. The reaction mixtures were boiled and analysed by SDS – PAGE and autoradiography.

In vitro interaction assays

Five hundred μg of cellular proteins from untreated or anti-Fas-treated Jurkat cells was incubated with 20 μg of purified GST-Crk (or GST-Grb2) proteins and 25 μl packed GSH-conjugated agarose beads in 1.5 ml of the lysis buffer at 4°C for 2 h with continuous rotation. The affinity-purified complex were then washed three times with 1.5 ml of the lysis buffer, denatured by adding 70 μl of 1×SDS sampling buffer and 5-min boiling, and analysed by Western blot assays using anti-HPK1 antibodies.

Western blot analyses

For Western blot analyses, the samples were prepared as described above. The samples were resolved by SDS – PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with a primary antibody (anti-HPK1 [Ab2025, Ab484, or Ab1404], 1 : 1000 dilution), washed, and blotted with a secondary antibody conjugated with HRP (1 : 1000 dilution). The membrane was then developed in the ECL reagent (Amersham) and exposed to an X-ray film.

Abbreviations

Crk:

CT10 regulator of kinase

Grb2:

growth factor receptor bound protein 2

GST:

glutathione S-transferase

HPK1:

hematopoietic progenitor kinase 1

JNK:

c-Jun N-terminal kinase

kD:

kilodalton

MAPK:

mitogen-activated protein kinase

MBP:

myelin basic protein

MEKK1:

MAPK kinase kinase 1

MKK:

MAPK kinase

PAK:

p21-GTPase-activated kinases

z-DEVD-FK:

z-Asp-Glu-Val-Asp-fluoromethyl ketone

z-VAD-FK:

z-Val-Ala-Asp-FK

References

  1. Anafi M, Kiefer F, Gish GD, Mbamalu G, Iscove NN and Pawson T. . 1997 J. Biol. Chem. 272: 27804–27811.

  2. Bagrodia S, Derijard B, Davis RJ and Ceroine RA. . 1995 J. Biol. Chem. 270: 27995–27998.

  3. Brenner B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F and Gulbins E. . 1997 J. Biol. Chem. 272: 22173–22181.

  4. Cardone M, Salvesen GS, Widmann C, Johnson G and Frisch SM. . 1997 Cell 90: 315–323.

  5. Chen Y-R, Meyer CF and Tan T-H. . 1996a J. Biol. Chem. 271: 631–634.

  6. Chen Y-R, Wang W, Kong A-NT and Tan T-H. . 1998 J. Biol. Chem. 273: 1769–1775.

  7. Chen Y-R, Wang X, Templeton D, Davis RJ and Tan T-H. . 1996b J. Biol. Chem. 271: 31929–31936.

  8. Deak JC, Cross JV, Lewis M, Qian Y, Parrott LA, Distelhorst CW and Templeton DJ. . 1998 Proc. Natl. Acad. Sci. USA 95: 5595–5600.

  9. Diener K, Wang XS, Chen C, Meyer CF, Bray J, Zukowski M, Tan T-H and Yao Z. . 1997 Proc. Natl. Acad. Sci. USA 94: 9687–9692.

  10. Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbabda S, Robertson M, Ghayur T, Wong WW, Kamen R, Weichselbaum R and Kufe D. . 1995 EMBO J. 14: 6148–6156.

  11. Erhardt P, Tomaselli KJ and Cooper GM. . 1997 J. Biol. Chem. 272: 15049–15052.

  12. Fanger GR, Gerwins P, Widmann C, Jarpe MB and Johnson GL. . 1997 Curr. Opin. Genet. Dev. 7: 67–74.

  13. Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E and Sanchez I. . 1997 Proc. Natl. Acad. Sci. USA 94: 3302–3307.

  14. Graves JD, Gotoh Y, Draves K, Ambrose D, Han DKM, Wright M, Chernoff J, Clark EA and Krebs EG. . 1998 EMBO J. 17: 2224–2234.

  15. Hu MC-T, Qiu WR, Wang X, Meyer CF and Tan T-H. . 1996 Genes & Dev. 10: 2251–2264.

  16. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K and Gotoh Y. . 1997 Science 275: 90–94.

  17. Ip YT and Davis RJ. . 1998 Curr. Opin. Cell Biol. 10: 205–219.

  18. Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A and Green DR. . 1998 Mol. Cell 1: 543–551.

  19. Kiefer F, Tibbles LA, Anafi M, Janssen A, Zanke BW, Lassam N, Pawson T, Woodgett JR and Iscove NN. . 1996 EMBO J. 15: 7013–7025.

  20. Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ and Williams LT. . 1997 Science 278: 294–298.

  21. Kyriakis JM and Avruch J. . 1996 J. Biol. Chem. 271: 24313–24316.

  22. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG and Earnshaw WC. . 1994 Nature 371: 346–347.

  23. Lazebnik YA, Takahashi RD, Moir RD, Goldman RD, Poirier GG, Kaufmann SH and Earnshaw WC. . 1995 Proc. Natl. Acad. Sci. USA 92: 9042–9046.

  24. Lenczowski JM, Lourdes D, Eder AM, King LB, Zacharchuk CM and Ashwell JD. . 1997 Mol. Cell. Biol. 17: 170–181.

  25. Levkau B, Koyama H, Raines EW, Clurman BE, Herren B, Orth K and Roberts JM. . 1998 Mol. Cell 1: 553–563.

  26. Ling P, Yao Z, Meyer CF, Wang XS, Oehrl W, Feller SM and Tan T-H. . 1999 Mol. Cell. Biol. 19: 1359–1368.

  27. Meyer CF, Wang X, Chang C and Tan T-H. . 1996 J. Biol. Chem. 271: 8971–8976.

  28. Oehrl W, Kardinal C, Ruf S, Adermann K, Groffen J, Feng G-S, Blenis J, Tan T-H and Feller SM. . 1998 Oncogene 17: 1893–1902.

  29. Park DS, Stefanis L, Irene Yan CY, Farinelli SE and Greene LA. . 1996 J. Biol. Chem. 271: 21898–21905.

  30. Pombo CM, Kehrl JH, Irma S, Woodgett JR, Force T and Kyriakis JM. . 1995 Nature 377: 750–754.

  31. Rehemtulla A, Hamilton CA, Chinnaiyan AM and Dixit VM. . 1997 J. Biol. Chem. 272: 25783–25786.

  32. Rudel T and Bokoch GM. . 1997 Science 276: 1571–1574.

  33. Salvesen G and Dixit VM. . 1997 Cell 91: 443–446.

  34. Seimiya H, Mashima T, Toha M and Tsuruo T. . 1997 J. Biol. Chem. 272: 4631–4636.

  35. Shiah S-G, Chuang S-E, Chau Y-P, Shen S-C and Kuo M-L. . 1998 Cancer Res. 59: 391–398.

  36. Song O, Lees-Miller SP, Kumar S, Zhang N, Chan DW, Smith GCM, Jackson SP, Alnemri ES, Litwack G, Khanna KK and Lavin MF. . 1996 EMBO J. 15: 3238–3246.

  37. Steller H. . 1995 Science 267: 1445–1449.

  38. Thompson CB. . 1995 Science 267: 1456–1462.

  39. Tournier C, Whitmarsh AJ, Vavanagh J, Barret T and Davis RJ. . 1997 Proc. Natl. Acad. Sci. USA 94: 7337–7342.

  40. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z and Kolesnick RN. . 1996 Nature 380: 75–79.

  41. Wang W, Zhou G, Hu MC-T, Yao Z and Tan T-H. . 1997 J. Biol. Chem. 272: 22771–22775.

  42. Wen L-P, Fahrni JA, Troie S, Guan J-L, Orth K and Rosen GD. . 1997 J. Biol. Chem. 272: 26056–26061.

  43. Widmann C, Gerwins P, Johson NL, Jarpe MB and Johnson GL. . 1998 Mol. Cell. Biol. 18: 2416–2429.

  44. Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg ME. . 1995 Science 270: 1326–1331.

  45. Yao Z, Brown A, Wang XS, Zhou G, Diener K, Zukowski M and Tan T-H. . 1997a J. Biol. Chem. 272: 2118–2125.

  46. Yao Z, Diener K, Wang XS, Zukowski M, Matsumoto G, Zhou G, Tibbles LA, Sasaki T, Nishina H, Tan T-H, Woodgett J and Penninger JM. . 1997b J. Biol. Chem. 272: 32378–32383.

  47. Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu F-F and Woodgett JR. . 1996 Curr. Biol. 6: 606–613.

  48. Zhou G, Lee SC, Yao Z and Tan T-H. . 1999 J. Biol. Chem. 274: 13133–13138.

Download references

Acknowledgements

We thank Drs SM Feller, MC-T Hu, and EY Skolnik for generous gifts, members of Tan laboratory for their helpful discussions and critical reading of this manuscript, S Lee and R Afshar for technical assistance, and M Lowe for secretarial assistance. This work was supported by the National Institutes of Health grants R01-AI38649 and R01-AI42532 (to T-H Tan). T-H Tan is a Scholar of the Leukemia Society of America. Y-R Chen was supported by a Department of Defense Predoctoral Fellowship (DAMD17-97-1-7078) in the Breast Cancer Research Program, and is a recipient of Department of Defense Postdoctoral Fellowship (DAMD17-99-1-9507) in the Prostate Cancer Research Program. CF Meyer was supported by an NIH postdoctoral Trainingship in Immunology.

Author information

Correspondence to Tse-Hua Tan.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chen, Y., Meyer, C., Ahmed, B. et al. Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1). Oncogene 18, 7370–7377 (1999) doi:10.1038/sj.onc.1203116

Download citation

Keywords

  • HPK1
  • JNK
  • caspase
  • apoptosis
  • adaptor

Further reading