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Originally published In Press as doi:10.1074/jbc.M410317200 on September 13, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49551-49561, November 19, 2004
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Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1*

Guisheng Zhou, Jonathan S. Boomer, and Tse-Hua Tan{ddagger}

From the Department of Immunology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, September 8, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic specific mammalian Ste20-like protein kinase and has been implicated in many cellular signaling pathways including T cell receptor (TCR) signaling. However, little is known about the in vivo regulation of HPK1. We present evidence that HPK1 is positively regulated by protein phosphatase 4 (PP4; also called PPX and PPP4), a serine/threonine phosphatase. We found that PP4 interacted with HPK1 and that the proline-rich region of HPK1 was necessary and sufficient for this interaction. We also found that PP4 had phosphatase activity toward HPK1 in vivo and that co-transfection of PP4 with HPK1 resulted in specific kinase activation of HPK1. Moreover, we found that the PP4-induced HPK1 kinase activation was accompanied by an increase in protein expression of HPK1. Pulse-chase analysis showed that PP4 increased the half-life of HPK1. Further studies showed that HPK1 was subject to regulation by ubiquitination and ubiquitin-targeted degradation and that PP4 inhibited HPK1 ubiquitination. In addition, we found that TCR stimulation enhanced the PP4-HPK1 interaction and that wild-type PP4 enhanced, whereas a phosphatase-dead PP4 mutant inhibited, TCR-induced activation of HPK1 in Jurkat T cells. Combined with the observation that PP4 enhanced HPK1-induced JNK activation, our studies identify PP4 as a positive regulator for HPK1 and the HPK1-JNK signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hematopoietic progenitor kinase 1 (HPK11; also named MAP4K1) belongs to the HPK1/GCk subgroup of mammalian Ste20-like kinases that specifically activate the JNK pathway and is considered as a potential MAPK kinase kinase kinase (1, 2). The HPK1/GCk subgroup of kinases also includes germinal center kinase (GCK), GCK-like kinase (GLK), HPK/GCK-like kinase/NcK-interacting kinase, and kinase homologous to Ste20/Sps1/GCK-related (1). These kinases are characterized by an N-terminal kinase domain, a C-terminal regulatory region, and the lack of a Rac1/Cdc42-binding domain found in p21-activated kinases, the other subgroup of mammalian Ste20-like kinases (1, 3). HPK1 is unique in that its expression is restricted to hematopoietic tissues of adults, although it is widely expressed in embryos (4, 5). It has been shown that HPK1 is involved in a variety of signaling systems (6), including epidermal growth factor (7, 8), transforming growth factor-{beta} (9, 10), erythropoietin (11), prostaglandin E2 (12), and T cell receptor (TCR) and B cell receptor stimulation (13–18). HPK1 is also involved in Fas ligation-mediated apoptosis (19, 20) and NF-{kappa}B activation (16, 21, 22). During TCR and B cell receptor signaling, HPK1 forms inducible complexes with a number of adaptor proteins, including Nck (15), Crk (15), the linker for activation of T cells (15, 18), the B cell adaptor containing Src homology 2 domain (also called BLNK or SLP-65) (16, 18), Clnk (17), SLP-76 (18), and Grb2-related adaptor downstream of Shc (also called Grap 2) (13, 23, 24). HPK1 also constitutively interacts with a variety of adaptor proteins such as Grb2 (7, 8, 14, 15), CrkL (8, 15, 25), HPK1-interacting protein of 55 kDa (also called SH3P7 and mAbp1) (26, 27), and Grap (14). We have shown that the adaptor protein, the HPK1-interacting protein of 55 kDa, is involved in the kinase activation of HPK1 by TCR stimulation (28). A recent phage display analysis found that HPK1 interacts with the cytoplasmic tail of membrane immunoglobulins (29). Although phosphorylation (4, 5, 7, 15–18, 30) and caspase-mediated cleavage (19, 22) have been implicated in the regulation of HPK1, the details of the in vivo regulation of HPK1 remain largely unknown.

Protein phosphatase 4 (PP4; also called PPX and PPP4) is a protein serine/threonine phosphatase that is structurally related to the PP2A family of phosphatases (31, 32). PP4 has been highly conserved over evolution, with human and Drosophila PP4 sharing 91% amino acid identity (31). Like PP2A, PP4 is a holoenzyme composed of catalytic (C), structural (A), and regulatory (B) subunits. To date, three PP4 subunits have been identified: {alpha}4, PP4-R1, and PP4-R2 (33–37). PP4 contains a putative binding domain for okadaic acid, a potent tumor promoter toxin (38). PP4 localizes to centrosomes in mammalian cells and in Drosophila embryos, and PP4 is involved in the regulation of microtubule growth or organization at centrosomes (36, 39–41). It has been recently found that PP4 plays a proapoptotic role in T lymphocytes (42). PP4 interacts with the survival of motor neurons complex and enhances the temporal localization of small nuclear ribonucleoprotein (43). Our previous studies show that PP4 interacts with components of NF-{kappa}B (e.g. c-Rel, p50, and RelA), stimulates the DNA binding activity of c-Rel, and activates NF-{kappa}B-mediated transcription (44). Recent studies demonstrate that PP4 dephosphorylates RelA (NF-{kappa}B p65), primarily on Thr-435, and that this dephosphorylation is required for NF-{kappa}B activation induced by cisplatin (45). We have recently shown that PP4 takes part in relaying a TNF-{alpha} signal to the activation of the JNK pathway (46) and down-regulation of insulin receptor substrate 4 (47). In our effort to investigate the molecular mechanism underlying the positive regulation of the JNK pathway by PP4, we found that PP4 interacted with and stabilized HPK1, leading to increased kinase activation of HPK1. In addition, PP4 enhanced the HPK1-induced JNK activation. We also observed that TCR stimulation enhanced the PP4-HPK1 interaction and that PP4 was involved in the kinase activation of HPK1 by TCR stimulation. These observations suggest that PP4 is a positive regulator for HPK1 and the HPK1-JNK signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—[{gamma}-32P]ATP, [32P]orthophosphate, and a mixture of [35S]methionine/cysteine were purchased from ICN Biomedicals (Irvine, CA). An ECL system was purchased from Amersham Biosciences. TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). Anti-HA antibody (12CA5) was purchased from Amersham Biosciences. Anti-FLAG (M2) and anti-{beta}-actin were purchased from Sigma. Polyclonal anti-HPK1 (N-19), monoclonal anti-PP1, and anti-c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-JNK1 antibody (Ab 101) (48), polyclonal anti-HPK1 antibody (Ab 484) (4, 5), anti-CD3 antibody (28), and polyclonal anti-PP4 antibody (Ab 104) (46) were previously described. All other chemical reagents were purchased from Sigma unless otherwise noted.

Plasmids—The glutathione S-transferase-Jun-(1–79) was a gift from Dr. M. Karin (University of California, San Diego). pHA-JNK1 was a gift from Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada). pHA-ubiquitin was a gift from Dr. X.-H. Feng (Baylor College of Medicine, Houston, TX). pCMV-PP1 was a gift from Dr. A. H. Schonthal (University of Southern California) (49). pMTSM-Myc-M3/6 was a gift from Dr. K. E. Davis (Oxford University) (50). FLAG-HPK1, FLAG-HPK1-M46, FLAG-HPK1-KD, FLAG-HPK1-CD, HA-HPK1-PR, and HA-HPK1-DR (8), PP4, HA-PP4, and HA-PP4-RL (44), and FLAG-GLK (51) were previously described.

Cells and Transfection—Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 units/ml streptomycin/penicillin. Human embryonic kidney 293T (HEK293T) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 100 units/ml streptomycin/penicillin. HEK293T cells were plated at a density of either 1.5 x 105 cells/35-mm well or 1.5 x 106 cells/100-mm dish and transfected the next day using the modified calcium phosphate precipitation protocol (Specialty Media, Inc., Lavallette, NJ). Cells were transfected with plasmids encoding {beta}-galactosidase (0.15 µg) in combination with an empty vector or various amounts of plasmids encoding phosphatases, phosphatase mutants, kinases, or kinase mutants as indicated in the figure legends to monitor the transfection efficiency. For those multiple transfections with identical component(s) (e.g. FLAG-HPK1 or FLAG-HPK1 plus PP4 in Fig. 6 and FLAG-HPK1 in Fig. 7A), we controlled transfection efficiency by performing FLAG-HPK1 transfection in 100-mm dishes and pooling the cells and aliquoting them into 6-well plates the next day after changing the medium. Jurkat cells (2 x 107/0.5 ml) were transiently transfected by electroporation (263 V, two pulses, and 10 ms) using a BTX Electro Square Porter T 820 (San Diego, CA). After electroporation, the cells were pooled (4 x 106/ml) and incubated overnight in RPMI 1640 supplemented with 5% fetal bovine serum. The cells were washed and resuspended in plain RPMI 1640 at a cell density of 1 x 108 cells/ml. 100 µl of the cell suspension (1 x 107) was aliquoted to tubes and incubated with 1 µl of anti-CD3 (OKT3) ascites for 10 min on ice. Goat anti-mouse antibody was added to cross-link the anti-CD3 (OKT3) ascites for 10 min with gentle rocking at 4 °C. Cell stimulations were performed at 37 °C for various time periods as indicated in the figure legend.



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  		FIG. 6.
PP4 increases the half-life of HPK1. HEK293T cells (1.5 x 105 cells in a 35-mm well) were transfected with FLAG-HPK1 (0.2 µg) alone (A) or FLAG-HPK1 (0.2 µg) plus PP4 (2 µg) (B). 36 h after transfection, the cells were starved in DMEM without methionine and cysteine for 1 h and then metabolically labeled with [35S]methionine/cysteine for 4 h. HEK293T cells were then chased in a nonradioactive medium for the indicated time period. Cells were lysed, and FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2) and analyzed by SDS-PAGE and autoradiography (upper panels). Densitometric analysis of FLAG-HPK1 was done using Kodak 1D Image Analysis Software (lower panels). The amount of FLAG-HPK1 at chasing time 0 h was set equal to 100%.

 



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  		FIG. 7.
HPK1 is subject to ubiquitin-targeted degradation. A, LLnL stabilizes HPK1. HEK293T cells (1.5 x 105 cells in 35-mm wells) were transfected with 0.5 µg of FLAG-HPK1. After 24 h, the cells were treated with various concentrations of LLnL as indicated for 16 h. The cell lysate was prepared and subjected to Western blot analysis with an anti-FLAG antibody (M2) (top). The blot was then reprobed with an anti-{beta}-actin antibody (bottom). B, HPK1 is ubiquitinated. HEK293T cells (1.5 x 106 cells in 100-mm dishes) were transfected with vector alone, HA-ubiquitin (0.2 µg) alone, FLAG-HPK1 (5 µg) alone, or FLAG-HPK1 (5 µg) plus HA-ubiquitin (0.2 µg) with or without treatment of LLnL (20 µM) for 16 h. The cells were collected 40 h post-transfection. FLAG-HPK1 was immunoprecipitated (IP) with an anti-FLAG antibody (M2). The immunoprecipitates were then subjected to Western blotting (WB) using an anti-HA antibody (12CA5) (upper panel). The expression of FLAG-HPK1 was monitored by Western blot analysis with an anti-FLAG antibody (M2) (lower panel). C, PP4 inhibits HPK1 ubiquitination. HEK293T cells (1.5 x 106 cells in 100-mm dishes) were co-transfected with FLAG-HPK1 (5 µg) plus PP4 (10 µg) or PP4-RL (10 µg) in the presence of HA-ubiquitin (0.2 µg). The cells were collected 40 h post-transfection. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2). The immunoprecipitates were then subjected to Western blotting using an anti-HA antibody (12CA5) (upper panel). The expression levels of FLAG-HPK1, PP4, and PP4-RL were monitored by Western blot analysis with an anti-FLAG antibody (M2) and an anti-PP4 antibody (Ab 104), respectively (lower panels).

 
Coimmunoprecipitation, Immunocomplex Kinase Assays, and Western Blot Analysis—Coimmunoprecipitation and immunocomplex kinase assays were performed as previously described (9, 10, 52). Western blot analysis was performed using an ECL kit according to the manufacturer's protocols (Amersham Biosciences).

Labeling HPK1 in Vivo—HEK293T cells (1.5 x 106 cells in 100-mm dishes) were transfected with vector, FLAG-HPK1 (10 µg) alone, FLAG-HPK1 (10 µg) plus PP4 (10 µg), or FLAG-HPK1 (10 µg) plus PP4-RL (10 µg). Empty vector was used to normalize the amount of transfected DNA. 40 h post-transfection, the cells were maintained in the serum-free, phosphate-free DMEM for 2 h at 37 °C. The cells were then labeled in the phosphate-free DMEM supplemented with 10% dialyzed serum and 100 µCi of [32P]orthophosphate/ml for 4 h at 37 °C. The cells were washed with PBS twice to remove free [32P]orthophosphate. FLAG-HPK1 was immunoprecipitated with an anti-FLAG (M2) antibody and subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride and autoradiographed. The polyvinylidene difluoride membrane was then subjected to immunoblotting using an anti-FLAG (M2) antibody.

Pulse-Chase Analysis—Pulse-chase experiments were performed using the [35S]methionine/cysteine mixture to monitor changes in the half-life of HPK1 in the presence or the absence of PP4. Briefly, 1.5 x 105 HEK293T cells/35-mm well were transfected with FLAG-HPK1 (0.2 µg) alone or with FLAG-HPK1 (0.2 µg) plus PP4 (2 µg). 36 h after transfection, the cells were starved in DMEM without methionine and cysteine for 1 h and then metabolically labeled with [35S]methionine/cysteine for 4 h. HEK293T cells were then chased in nonradioactive medium for the time periods as indicated. Cells were lysed, and the cell samples containing equal amounts of proteins were immunoprecipitated with an anti-FLAG antibody (M2). Immunocomplexes were collected with immobilized protein G-Sepharose beads and resolved on SDS-PAGE. The gels were dried and autoradiographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HPK1 Interacts with PP4 through Its Proline-rich Region—We have previously found that PP4 acts as a positive regulator for the JNK pathway during TNF-{alpha} signaling and that PP4 probably exerts its effect on JNK in an indirect manner, since no direct PP4-JNK interaction was detected (46). The JNK pathway is composed of multiple kinases (53, 54). In our effort to explore the molecular mechanism underlying the positive regulation of the JNK pathway by PP4, we sought to identify the target(s) of PP4 within the JNK pathway. We co-transfected PP4 into HEK293T cells with an individual upstream activating kinase of the JNK pathway, including MAPK kinases, MAPK kinase kinases, and MAPK kinase kinase kinases, followed by immunoprecipitation/Western blotting. We found that HPK1 is one of the upstream activating kinases that interacted with PP4. As shown in Fig. 1, FLAG-HPK1 was co-immunoprecipitated with PP4 when a specific anti-PP4 antibody (46) was used to immunoprecipitate PP4 (Fig. 1A, left panel). Conversely, HA-PP4 was co-immunoprecipitated with FLAG-HPK1 when FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (Fig. 1A, right panel). To exclude potential artificial interaction due to the overexpression system, we investigated whether endogenous PP4 and HPK1 interact with each other in Jurkat T cells, a cell line that abundantly expresses both HPK1 and PP4. We found that endogenous HPK1 was co-immunoprecipitated with endogenous PP4 when PP4 was immunoprecipitated with an anti-PP4 antibody (Fig. 1B). Taken together, these data suggest that HPK1 is a PP4-interacting protein and a potential PP4 target within the JNK pathway.



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  		FIG. 1.
HPK1 interacts with PP4. A, HPK1 interacts with PP4 in transiently transfected HEK293T cells. HEK293T cells (1 x 106 cells in a 100-mm dish) were transfected with PP4 (10 µg), FLAG-HPK1 (10 µg), or FLAG-HPK1 (10 µg) plus PP4 (10 µg) (left). HEK293T cells (1 x 106 cells in a 100-mm dish) were transfected with FLAG-HPK1 (5 µg), HA-PP4 (5 µg), or FLAG-HPK1 (5 µg) plus HA-PP4 (5 µg) (right). Empty vector was used to normalize the amount of transfected DNA. 42 h post-transfection, cell lysates were prepared. PP4 was immunoprecipitated (IP) with an anti-PP4 antibody (Ab 104). The immunoprecipitates were then immunoblotted (WB) with an anti-FLAG antibody (M2) (left). Conversely, FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2), and the immunoprecipitates were immunoblotted with an anti-HA antibody (12CA5) (right). The expression levels of PP4, HA-PP4, and FLAG-HPK1 were monitored by immunoblotting using an anti-PP4 antibody (Ab 104), an anti-HA antibody (12CA5), and an anti-FLAG antibody (M2), respectively (lower panels). B, PP4 interacts with HPK1 in Jurkat T cells. PP4 from 1.5 x 108 Jurkat T cells was immunoprecipitated with an anti-PP4 antibody (Ab 104). The immunoprecipitate was then immunoblotted with an anti-HPK1 antibody (N-19).

 
To further understand the PP4-HPK1 interaction, we mapped the region within HPK1 that is required for this interaction. We generated four truncated forms of HPK1, including the N-terminal kinase domain of HPK1 (HPK1-KD; amino acids 1–291), the C-terminal regulatory domain of HPK1 (HPK1-CD; amino acids 292–833), the proline-rich region of HPK1 (HPK1-PR; amino acids 288–482), and the distal region of HPK1 (HPK1-DR; amino acids 483–833) (Fig. 2A). We first co-transfected HA-PP4 into HEK293T cells with either HPK1-KD or HPK1-CD. Immunoprecipitation/Western blot analysis showed that only HPK1-CD (Fig. 2B, right panel), and not HPK1-KD (Fig. 2B, left panel), associated with PP4 when HA-PP4 was immunoprecipitated with an anti-HA antibody. These data indicate that the region involved in the PP4-HPK1 interaction resides in the C-terminal regulatory domain of HPK1. To narrow the region required for the PP4-HPK1 interaction, we divided the C-terminal regulatory domain of HPK1 into the proline-rich region (HPK1-PR) and the distal region (HPK1-DR) containing a citron homology domain (Fig. 2A). Next, we co-transfected HPK1-PR and HPK1-DR into HEK293T cells with PP4 and FLAG-PP4, respectively. Immunoprecipitation/Western blot analysis showed that only HPK1-PR (Fig. 2C, left panel), and not HPK1-DR (Fig. 2C, right panel), associated with PP4. These data indicate that the proline-rich region of HPK1 is necessary and sufficient for the PP4-HPK1 interaction. Therefore, our mapping studies identified the proline-rich region of HPK1 as the potential domain involved in the PP4-HPK1 interaction.



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  		FIG. 2.
The proline-rich region of HPK1 mediates the PP4-HPK1 interaction. A, a schematic representation of HPK1 and its four truncated forms, including the kinase domain of HPK1 (HPK1-KD; amino acids 1–291), the C-terminal regulatory domain of HPK1 (HPK1-CD; amino acids 292–833), the proline-rich region of HPK1 (HPK1-PR; amino acids 288–482), and the distal region of HPK1 (HPK1-DR; amino acids 483–833). HPK1-KD and HPK1-CD were FLAG-tagged, whereas HPK-PR and HPK1-DR were HA-tagged. B, HPK1-CD, but not HPK1-KD, interacts with PP4. HEK293T cells (1.5 x 106 cells in a 100-mm dish) were transfected with HA-PP4 (5 µg) alone, HA-PP4 (5 µg) plus FLAG-HPK1-KD (5 µg) (left), or HA-PP4 (5 µg) plus FLAG-HPK1-CD (5 µg) (right). Empty vector was used to normalize the amount of transfected DNA. 42 h post-transfection, cell lysates were prepared. HA-PP4 was immunoprecipitated (IP) with an anti-HA antibody (12CA5). The immunoprecipitates were then immunoblotted (WB) with an anti-HPK1 antibody (N-19) for HPK1-KD, and an anti-HPK1 antibody (Ab 484) for HPK1-CD (top of the panels). The expression levels of HA-PP4, FLAG-HPK1-KD, and FLAG-HPK1-CD were monitored by immunoblotting using an anti-HA antibody (12CA5), an anti-HPK1 antibody (N-19), and an anti-HPK1 antibody (Ab 484), respectively (lower panels). C, HPK1-PR, but not HPK1-DR, interacts with PP4. HEK293T cells (1 x 106 cells in a 100-mm dish) were transfected with PP4 (10 µg) alone or PP4 (10 µg) plus HA-HPK1-PR (10 µg) (left) or with FLAG-PP4 (5 µg) alone or FLAG-PP4 (5 µg) plus HA-HPK1-DR (5 µg) (right). Empty vector was used to normalize the amount of transfected DNA. 42 h post-transfection, cell lysates were prepared. PP4 and FLAG-PP4 were immunoprecipitated with an anti-PP4 antibody and an anti-FLAG antibody (M2), respectively. The immunoprecipitates were then immunoblotted with an anti-HA antibody (12CA5) for HA-HPK1-PR and HA-HPK1-DR (top of the panels). The expression levels of PP4, FLAG-PP4, HA-HPK1-PR, and HA-HPK1-DR were monitored by immunoblotting using an anti-PP4 (Ab 104) antibody, an anti-FLAG (M2) antibody, and an anti-HA (12CA5) antibody, respectively (lower panels).

 
PP4 Dephosphorylates HPK1—PP4 is a serine/threonine phosphatase. HPK1 is subject to regulation by autophosphorylation on multiple serine and threonine residues.2 The physical interaction between PP4 and HPK1 suggests that HPK1 may be a substrate for PP4. To test this hypothesis, we examined whether PP4 has phosphatase activity toward HPK1. We cotransfected FLAG-HPK1 into HEK293T cells with PP4 or PP4-RL, a phosphatase-dead mutant of PP4 in which arginine 236 is replaced with leucine (46), and labeled the cells with [32P]orthophosphate. HPK1 was immunoprecipitated and subjected to SDS-PAGE analysis and autoradiography. We found that co-transfection of PP4 with HPK1 resulted in a significant decrease in the intensity of total HPK1 phosphorylation, whereas PP4-RL had no such effect (Fig. 3). These data indicate that PP4 has phosphatase activity toward HPK1 in vivo, and HPK1 is thus a potential substrate of PP4.



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  		FIG. 3.
PP4 dephosphorylates HPK1. HEK293T cells (1.5 x 106 cells in a 100-mm dish) were transfected with vector alone, FLAG-HPK1 (10 µg) alone, FLAG-HPK1 (10 µg) plus PP4 (10 µg), or FLAG-HPK1 (10 µg) plus PP4-RL (10 µg). 40 h post-transfection, the cells were maintained in the phosphate-free DMEM for 2 h at 37 °C. The cells were then labeled in the phosphate-free DMEM supplemented with 10% of dialyzed serum and 100 µCi of [32P]orthophosphate for 4 h at 37 °C. FLAG-HPK1 was immunoprecipitated (IP) with an anti-FLAG (M2) antibody and subjected to SDS-PAGE, transferal to polyvinylidene difluoride membrane, and autoradiography (top). The polyvinylidene difluoride membrane was then subjected to immunoblotting (WB) using an anti-FLAG antibody (M2) (bottom).

 
PP4 Is a Specific Positive Regulator of HPK1 and the HPK1-JNK Cascade—To explore the functional relevance of the PP4-HPK1 interaction, we examined the effect of PP4 on HPK1. We co-transfected HPK1 into HEK293T cells with PP4 and measured HPK1 kinase activity by performing immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. We found that co-transfection of PP4 with HPK1 resulted in kinase activation of HPK1 (Fig. 4A, top). Western blot analysis showed that PP4 increased the protein expression of HPK1 (Fig. 4A, bottom). Thus, PP4 acts as a positive regulator for both kinase activity and protein expression of HPK1. To determine the specificity of the positive regulation of HPK1 by PP4, we examined the effect of PP4 on GLK, another member of the HPK1/GCK subgroup of mammalian Ste20-like kinases (1, 3). In contrast to HPK1, PP4 did not up-regulate the protein expression level or kinase activity of GLK (Fig. 4A). To further determine the specificity of the effect of PP4 on HPK1, we examined the effect of PP1, a serine/threonine phosphatase, and M3/6, a dual specificity MAPK phosphatase, on HPK1. Neither PP1 nor M3/6 increased the protein expression or kinase activity of HPK1 (Fig. 4B). Taken together, these data indicate that PP4 is a specific positive regulator of HPK1.



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  		FIG. 4.
PP4 is a specific positive regulator of HPK1 and the HPK1-JNK cascade. A, PP4 only activates HPK1 and not GLK. 2 µg of PP4 was co-transfected into HEK293T cells (1.5 x 105 cells in a 35-mm well) with 100 ng of FLAG-HPK1 or FLAG-GLK. Empty vector was used to normalize the amount of transfected DNA. The cells were collected 42 h after transfection. FLAG-HPK1 and FLAG-GLK were immunoprecipitated (IP) with an anti-FLAG antibody (M2). Immunocomplex kinase assays were performed using MBP as a substrate (top panel). Expression levels of PP4, HPK1 and GLK were monitored by immunoblotting (WB) with an anti-PP4 antibody (Ab 104) and an anti-FLAG antibody (M2) (lower panels). B, HPK1 is specifically activated by PP4 and not by PP1 and M3/6. FLAG-tagged HPK1 (100 ng) was co-transfected into HEK293T cells (1.5 x 105 cells in a 35-mm well) with 2 µg of empty vector, PP4, PP1, or M3/6. Empty vector was used to normalize the amount of transfected DNA. The cells were collected 42 h after transfection. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2). Immunocomplex kinase assays were performed using MBP as a substrate (top panel). Expression levels of PP4, PP1, Myc-M3/6, and FLAG-HPK1 were monitored by immunoblotting with an anti-PP4 (Ab 104), anti-PP1, anti-Myc, and anti-FLAG (M2) antibody, respectively (lower panels). C, PP4 enhances HPK1-induced JNK activation. HEK293T cells (1.5 x 105 cells in a 35-mm well) were transfected with HA-JNK1 (0.1 µg) alone, HA-JNK1 plus FLAG-HPK1 (2 µg), HA-JNK1 plus FLAG-HPK1 and HA-PP4-RL (2 µg), or HA-JNK1 plus FLAG-HPK1 and PP4 (2 µg). Empty vector was used to normalize the amount of transfected DNA. Cell lysates were prepared at 42 h post-transfection, HA-JNK1 was immunoprecipitated with an anti-HA antibody (12CA5), and immunocomplex kinase assays were done using glutathione S-transferase-c-Jun-(1–79) as a substrate. The expression levels of HA-JNK, FLAG-HPK1, PP4-RL, and PP4 were monitored by immunoblotting using an anti-HA (12CA5), an anti-FLAG (M2), and an anti-PP4 (Ab 104) antibody, respectively.

 
HPK1 is a known upstream activating kinase of the JNK pathway (4, 5). Given that PP4 activated HPK1, we expected that PP4 should enhance the kinase activation of JNK by HPK1. To test this hypothesis, we co-transfected HA-JNK1 into HEK293T cells with FLAG-HPK1 in the presence of wild-type PP4 or phosphatase-dead PP4 mutant (PP4-RL). JNK kinase activity was measured by immunocomplex kinase assays using glutathione S-transferase-c-Jun-(1–79) as a substrate. We found that wild-type PP4 enhanced HPK1-induced JNK activation (Fig. 4C, lane 4 versus lane 2), whereas phosphatase-dead PP4-RL inhibited HPK1-induced JNK activation (Fig. 4C, lane 3 versus lane 2). Our previous study showed that PP4 positively regulates the JNK kinase activity in an indirect manner, since no direct PP4-JNK interaction was detected (46). Given that PP4 interacted with HPK1 (Figs. 1 and 2) and that PP4 had phosphatase activity toward HPK1 (Fig. 3), it is likely that PP4 positively regulates the JNK pathway by activating HPK1.

PP4 Activates HPK1 and Increases Protein Expression of HPK1—To further understand the positive regulation of HPK1 by PP4, we co-transfected HPK1 into HEK293T cells with increasing amounts of PP4. We found that co-transfection of PP4 with HPK1 resulted in the kinase activation of HPK1 in a PP4 concentration-dependent manner (Fig. 5A, top). Western blot analysis showed that PP4 increased the protein expression of HPK1 (Fig. 3A, bottom). Moreover, it seems that PP4 increased the protein expression and kinase activity of HPK1 through two distinct mechanisms, since the kinase activity of HPK1 increased before its protein levels increased (Fig. 5A, lane 4 versus lane 2).



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  		FIG. 5.
PP4 activates HPK1 and increases protein expression of HPK1. A, HPK1 protein expression and kinase activity are increased by PP4 in a dose-dependent manner. FLAG-HPK1 (50 ng) was co-transfected into HEK293T cells (1.5 x 105 cells in a 35-mm well) with various amounts of PP4 in a range from 0.1 to 2 µg. Empty vector was used to normalize the amount of transfected DNA. Cell lysates were prepared at 42 h post-transfection. FLAG-HPK1 was immunoprecipitated (IP) with an anti-FLAG antibody (M2), and immunocomplex kinase assays were performed using MBP as a substrate. The expression levels of FLAG-HPK1 and PP4 were monitored by immunoblotting (WB) using an anti-FLAG (M2) and an anti-PP4 (Ab 104) antibody, respectively. B, TNF-{alpha} enhances PP4-induced activation of HPK1. HEK293T cells (1.5 x 105 cells in a 35-mm well) were transfected with FLAG-HPK1 (0.1 µg) alone, FLAG-HPK1 (0.1 µg) plus PP4 (2 µg), or FLAG-HPK1 (0.1 µg) plus HA-PP4-RL (2 µg). Empty vector was used to normalize the amount of transfected DNA. 40 h post-transfection, the cells were treated with TNF-{alpha} (10 ng/ml) for various time periods as indicated. Cell lysates were prepared. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2), and immunocomplex kinase assays were performed using MBP as a substrate. Expression levels of FLAG-HPK1 and PP4/HA-PP4-RL were monitored by immunoblotting using an anti-FLAG (M2) and an anti-PP4 antibody, respectively.

 
TNF-{alpha} is a stimulatory cytokine for PP4 (46). We co-transfected HPK1 into HEK293T cells with PP4 or PP4-RL, followed by TNF-{alpha} treatment. We found that co-transfection of wild-type PP4 with HPK1 resulted in activation of HPK1 (Fig. 5B, top, lane 2), whereas PP4-RL had no effect on HPK1 (Fig. 5B, top, lane 3). As expected, HPK1 expression increased only in the presence of wild-type PP4 (Fig. 5B, bottom, lane 2) and not mutant PP4 (Fig. 5B, bottom, lane 3). These results indicate that PP4 phosphatase activity is required for its effect on HPK1. TNF-{alpha} treatment resulted in a time-dependent increase of HPK1 kinase activity in the presence of PP4 (Fig. 5B, lanes 2, 5, 8, 11, and 14). However, TNF-{alpha} had no effect on HPK1 in the presence of PP4-RL (Fig. 5B, lanes 3, 6, 9, 12, and 15). These data indicate that TNF-{alpha} exerted its effect on HPK1 through PP4. TNF-{alpha} treatment did not further increase HPK1 protein expression in the presence of PP4 (Fig. 5B, lanes 2, 5, 8, 11, and 14), suggesting that TNF-{alpha} up-regulated HPK1 through PP4-mediated modulation of its intrinsic kinase activity rather than its protein expression. Taken together, these data indicate that PP4 contributed to the up-regulation of both kinase activity and protein expression of HPK1. It should be noted that PP4 induced increases in both protein expression and kinase activity of HPK1 only when lower amounts of HPK1 were used in the transfection assays (data not shown). This is probably due to HPK1 being constitutively activated when overexpressed and to the kinase activity from large amounts of overexpressed HPK1 overriding the effect of PP4. To exclude the possibility that the enhanced protein expression of HPK1 is due to the PP4 activation of the cytomegalovirus promoter of the HPK1 expression construct, we compared the effect of PP4 on wild-type HPK1 and truncated HPK1-CD, the C-terminal regulatory domain of HPK1 (amino acids 292–833) (Fig. 2A). In contrast to wild-type HPK1, the protein expression of HPK1-CD was not affected by PP4 (data not shown). Therefore, PP4 exerted its effect directly on HPK1 itself rather than the cytomegalovirus promoter of the HPK1 expression construct.

PP4 Stabilizes HPK1—Next we addressed the underlying mechanism by which HPK1 is positively regulated by PP4. One possible mechanism is that PP4 stabilizes HPK1, thus increasing HPK1 protein expression. To test this hypothesis, we performed pulse-chase analyses to examine the effect of PP4 on the half-life of HPK1 protein. FLAG-HPK1 was co-transfected into HEK293T cells with wild-type PP4 or an empty vector, and the cells were labeled with [35S]methionine. After 4 h, the [35S]methionine-labeled cells were chased with unlabeled medium for various time periods up to 4 h. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2) and analyzed by SDS-PAGE and autoradiography (Fig. 6A). The half-life of HPK1 in the absence of PP4 was less than 2 h (Fig. 6B). Co-transfection of PP4 with HPK1 resulted in a significant increase of the half-life of HPK1 from less than 2 h to beyond 4 h (Fig. 6B). These data suggest that PP4 stabilizes HPK1.

HPK1 Is Subject to Ubiquitination and Ubiquitin-targeted Degradation—Ubiquitin-targeted degradation is one of the major mechanisms that control protein levels in vivo. Therefore, we examined whether HPK1 is subject to regulation by ubiquitination. We first examined the effect of the proteasome inhibitor N-acetyl-L-leucyl-leucyl-L-norleucinal (LLnL) on HPK1 protein expression. We found that LLnL enhanced HPK1 protein expression in a dose-dependant manner (Fig. 7A), indicating that HPK1 is subject to ubiquitin-targeted degradation. We noticed that LLnL-induced increase of HPK1 protein expression was accompanied by the appearance of FLAG-HPK1 doublets (Fig. 7A, lanes 6–8). The upper band(s) most likely represents autophosphorylated HPK1, since overexpressed HPK1 is constitutively activated in HEK293T cells and this activation is accompanied by autophosphorylation on serine and threonine residues.3 This phenomenon can be easily observed at higher concentrations of overexpressed HPK1. Therefore, we speculate that LLnL stabilizes HPK1, which was accompanied by increased autophosphorylation.

We next examined whether HPK1 is able to be ubiquitinated. We co-transfected HPK1 with HA-tagged ubiquitin into HEK293T cells. FLAG-HPK1 was immunoprecipitated with an anti-FLAG antibody (M2) and subjected to Western blotting with an anti-HA antibody. As shown in Fig. 7B, HPK1 was multiubiquitinated (lane 4). Treatment of the cells with LLnL increased both protein expression and ubiquitination of HPK1 (Fig. 7B, lane 5). Thus, HPK1 is a labile protein that is subject to degradation by the ubiquitin-directed proteasome complex. We then examined the effect of PP4 on HPK1 ubiquitination. We co-transfected HPK1 into HEK293T cells with wild-type PP4 or phosphatase-dead PP4-RL in the presence of HA-ubiquitin. While PP4-RL had no significant effect on HPK1 ubiquitination, wild-type PP4 inhibited HPK1 ubiquitination (Fig. 7C). Taken together, these data suggest that one mechanism for the positive regulation of HPK1 by PP4 is through inhibiting HPK1 ubiquitination and stabilizing HPK1.

Kinase Activity of HPK1 Is Required for the Action of PP4 on HPK1—Autophosphorylation has been implicated in the regulation of HPK1, although the details remain unknown. Since PP4 is a serine/threonine phosphatase and PP4 has phosphatase activity toward HPK1, we questioned whether the effect of PP4 on HPK1 is dependent on the autophosphorylation activity of HPK1. HPK1-M46 is a kinase-dead mutant, in which lysine 46 is replaced with methionine, resulting in the loss of ATP binding ability and HPK1 kinase activity (4, 5). We compared the effect of PP4 on wild-type HPK1 and the kinase-dead HPK1-M46. We found that PP4 only increased the protein expression of wild-type HPK1 and not kinase-dead HPK1 (Fig. 8A), indicating that the stabilization of HPK1 by PP4 depends on HPK1 kinase activity. We also found that the proteasome inhibitor LLnL only stabilized wild-type HPK1 and not HPK1-M46 (Fig. 8B). Taken together, these data suggest that autophosphorylation activity may contribute to the ubiquitination and subsequent degradation of HPK1, and that PP4 exerts its effect only on autophosphorylated HPK1. Therefore, kinase activity of HPK1 is required for the action of PP4 on HPK1.



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  		FIG. 8.
HPK1 kinase activity is required for its stabilization by PP4. A, PP4 only stabilizes HPK1 and not HPK1-M46. 0.2 µg of FLAG-HPK1 or FLAG-HPK1-M46 was co-transfected into HEK293T cells (1.5 x 105 cells in 35 mm wells) with various amounts (0.1, 1, and 2 µg) of PP4. The cells were collected 40 h post-transfection. The cell lysate was prepared and subjected to Western blot analysis with an anti-FLAG antibody (M2). B, LLnL stabilizes HPK1 but not HPK1-M46. HEK293T cells (1.5 x 105 cells in 35-mm wells) were transfected with 0.5 µg of FLAG-HPK1 or FLAG-HPK1-M46. After 24 h, the cells were treated with various concentrations of LLnL as indicated for 16 h. The cell lysate was prepared and subjected to Western blot analysis (WB) with an anti-FLAG antibody (M2).

 
PP4 Is Involved in HPK1 Activation by TCR Stimulation—It has been well established that HPK1 is involved in TCR signaling (6). To explore the physiological relevance of the PP4-HPK1 interaction, we examined whether PP4 is involved in HPK1 activation by TCR stimulation in Jurkat cells. We cotransfected FLAG-HPK1 into Jurkat T cells with HA-PP4. Immunoprecipitation/Western blotting analysis showed that TCR stimulation enhanced the PP4-HPK1 interaction in a time-dependent manner (Fig. 9A). We then cotransfected FLAG-HPK1 with either wild-type HA-PP4 or phosphatase-dead HA-PP4-RL into Jurkat T cells. After TCR stimulation, HPK1 kinase activity was measured by immunocomplex kinase assays using MBP as a substrate. We found that wild-type PP4 enhanced, whereas phosphatase-dead PP4-RL inhibited, HPK1 kinase activation by TCR stimulation (Fig. 9B). These data suggest that PP4 is involved in TCR signaling by interacting with HPK1 and enhancing HPK1 kinase activation. We noticed that co-transfection of PP4 with HPK1 did not increase the protein expression level of HPK1 in Jurkat T cells (Fig. 9), indicating that PP4-mediated HPK1 kinase activation by TCR signaling is not due to an increase in HPK1 protein levels.



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  		FIG. 9.
PP4 is involved in HPK1 activation by TCR stimulation. A, TCR stimulation enhances the PP4-HPK1 interaction. Jurkat T cells (2 x 107) were transfected with empty vector (40 µg) or HA-PP4 (20 µg) plus FLAG-HPK1 (20 µg) by electroporation. The transfected cells were then stimulated with anti-CD3 (OKT3) ascites for various time periods as indicated. After stimulation, cell lysates were prepared, and PP4 was immunoprecipitated (IP) by an anti-HA (12CA5) antibody. The immunoprecipitates were then subjected to Western blot (WB) using an anti-HPK1 (Ab 484) antibody for associated HPK1 or an anti-HA (12CA5) antibody for immunoprecipitated HA-PP4. B, PP4 enhances, whereas PP4-RL inhibits, TCR-induced HPK1 activation. Jurkat T cells (2 x 107) were transiently transfected by electroporation with DNA for vector control (25 µg) or FLAG-HPK1 (10 µg) in the presence or absence 15 µg of either HA-PP4 or HA-PP4-RL. Empty vector was used to normalize the amount of transfected DNA. After TCR stimulation for 5 min, cell lysates were prepared. FLAG-HPK1 was immunoprecipitated from 100 µg of cell lysate with an anti-FLAG (M2) antibody, and immunocomplex kinase assays were performed using MBP as a substrate. Expression levels of FLAG-HPK1 and HA-PP4/HA-PP4-RL were monitored by immunoblotting using an anti-HPK1 (Ab 484) and an anti-HA (12CA5) antibody, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mammalian Ste20-like kinases provide a potential link between the MAPK cascades and extracellular stimuli (1, 2). Moreover, some mammalian Ste20-like kinases are involved in MAPK-independent cellular functions (14, 55–59). The mammalian Ste20-like kinases are divided into the p21-activated kinase and HPK1/GCK subgroups. In comparison with the p21-activated kinase subgroup (60–64), much less is known about the cellular regulation of the HPK1/GCK subgroup of kinases. We provide evidence that HPK1 is positively regulated by PP4, a serine/threonine protein phosphatase. The evidence includes the following: (i) PP4 interacted with HPK1; (ii) PP4 showed an in vivo phosphatase activity toward HPK1; (iii) PP4 inhibited the ubiquitination of HPK1 and increased the half-life of HPK1, leading to the stabilization and activation of HPK1; (iv) PP4 enhanced HPK1-induced JNK activation; (v) PP4 inducibly associated with HPK1 during TCR signaling; and (vi) wild-type PP4 enhanced, whereas phosphatase-dead PP4 inhibited, HPK1 activation by TCR stimulation. Our studies thus identified PP4 as a potential positive regulator of HPK1 in TCR signaling.

Several signaling components of the MAPK pathways are subject to regulation by ubiquitination. These proteins include the {gamma}-p21-activated protein kinase ({gamma}-p21-activated kinase; a mammalian Ste20-like kinase) (65, 66), the apoptosis signal-regulated kinase 1 (ASK1; a MAPK kinase kinase) (67), Ste7 (a yeast MAPK kinase) (68, 69), extracellular signal-regulated kinases 1, 2, and 3 (70, 71), and the islet-brain 1/JNK-interacting protein 1 (a JNK scaffold protein) (72). Transforming growth factor-{beta}-activated kinase 1 (9, 10, 73) is another MAPK kinase kinase that is probably subject to regulation by ubiquitination (74). Our studies showed that PP4-induced HPK1 kinase activation was accompanied by a significant increase of HPK1 protein expression. Moreover, we found that the proteasome inhibitor LLnL increased ubiquitin-containing HPK1 and that PP4 inhibited HPK1 ubiquitination and increased the half-life of HPK1. Thus, we provide pharmacological and biochemical evidence that HPK1 is another signaling component of the JNK pathway that is subject to regulation by ubiquitination and that PP4 is involved in the regulation of HPK1 stability.

Multiple signaling components of the JNK pathway are negatively regulated by phosphorylation through different mechanisms. For example, the Ser-83 phosphorylation of ASK1, a MAPK kinase kinase, by AKT (75) and the Thr-131 phosphorylation of JNK3 by cyclin-dependent kinase 5 (76) inhibits ASK1 and JNK3 kinase activation, whereas the Ser-78 phosphorylation of SEK1/MKK4 by AKT inhibits the SEK1/MKK4-JNK interaction, leading to inhibition of the JNK signaling (77). It is reasonable to speculate that the corresponding phosphatases responsible for dephosphorylating these inhibitory residues will exert their stimulatory effect on the JNK pathway through different mechanisms. PP4 is a serine/threonine phosphatase. Given that PP4 physically interacted with HPK1 and that PP4 had phosphatase activity toward HPK1, one potential mechanism underlying the positive regulation of HPK1 by PP4 may be that PP4-mediated dephosphorylation leads to the stabilization and/or the kinase activation of HPK1. We observed that PP4 only stabilized wild-type HPK1 and not kinase-dead HPK1-M46, indicating that the HPK1 kinase activity is required for the regulation of its stability by PP4. Thus, we speculate that PP4 targets an autophosphorylated residue that inhibits HPK1 stability. In fact, for most unstable protein kinases, destabilization is linked to their enzymatic activation (78–81), although the control of extracellular signal-regulated kinase 3 stability is independent of its kinase activity (71). We also observed that the proteasome inhibitor LLnL only stabilized wild-type HPK1 and not kinase-dead HPK1-M46. Taken together, we hypothesize that HPK1 autophosphorylates itself and becomes susceptible to ubiquitination. PP4-mediated dephosphorylation blocks this phosphorylation-dependent ubiquitination and inhibits the subsequent ubiquitin-targeted degradation, resulting in the increased protein expression level of HPK1 and the subsequent increased kinase activation of HPK1. PP4-mediated deubiquitination and stabilization of HPK1 may contribute to the fine regulation of the strength and duration of HPK1 activation.

HPK1 activation by TCR and B cell receptor stimulation is a multistep event, including tyrosine phosphorylation, subcellular translocation, and the interaction with different adaptor proteins (13–18, 20, 23). Autophosphorylation has also been implicated in the kinase activation of HPK1 (4, 5). We have observed that HPK1 autophosphorylation occurs on multiple serine and threonine residues.2 Here, we showed that PP4 is involved in TCR-stimulated HPK1 kinase activation in Jurkat T cells, as indicated by the enhanced PP4-HPK1 interaction and the blockage of the HPK1 kinase activation by a phosphatase-dead PP4 mutant. However, we did not detect an increase in protein expression level of HPK1 by PP4, indicating that PP4-mediated HPK1 kinase activation by TCR signaling is not due to an increase in HPK1 protein levels. Instead, PP4 may directly modulate HPK1 kinase activity. We also observed that the PP4-stimulatory cytokine TNF-{alpha} enhances PP4-induced HPK1 kinase activation without further enhancing the effect of PP4 on HPK1 stabilization. These data indicate that, in addition to the stabilization of HPK1, PP4 may also exert its positive effect on HPK1 through other mechanisms. Identification of the PP4 dephosphorylation site(s) within HPK1 will help determine the molecular mechanism by which HPK1 is positively regulated by PP4.


   FOOTNOTES
 
* This work was supported by National Institutes of Health (NIH) Grants R01-CA87076 and R01-AI42532 (to T.-H. T.), Beginning Grant-in-Aid 0465156Y from the American Heart Association Texas Affiliate (to G. Z.), and NIH Grant 5-T32-A107495 (to J. S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Immunology, Baylor College of Medicine, One Baylor Plaza, M929, Houston, TX 77030. E-mail: ttan{at}bcm.tmc.edu.

1 The abbreviations used are: HPK1, hematopoietic progenitor kinase-1; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; GCK, germinal center kinase; GLK, GCK-like kinase; PP4, protein phosphatase 4; PP2A, protein phosphatase 2A; TNF-{alpha}, tumor necrosis factor {alpha}; HA, hemagglutinin; LLnL, N-acetyl-L-leucyl-leucyl-L-norleucinal; MBP, myelin basic protein; HPK1-KD, the kinase domain of HPK1; HPK1-CD, the C-terminal regulatory domain of HPK1; HPK1-PR, the proline-rich region of HPK1; HPK1-DR, the distal region of HPK1; TCR, T cell receptor; HEK293T, human embryonic kidney 293T; DMEM, Dulbecco's modified Eagle's medium; Ab, antibody. Back

2 G. Zhou, J. S. Boomer, and T.-H. Tan, unpublished results. Back

3 G. Zhou, J. S. Boomer, and T.-H. Tan, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank our colleagues for providing valuable reagents, members of the Tan laboratory for the helpful discussions and critical reading of the manuscript, M. Hu for technical assistance, and D. A. Guzman for administrative assistance.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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