Function and Regulation of HPK1 Family kinases in T-cell activation

附件一

行政院國家科學委員會補助專題研究計畫

X

成 果 報 告

□期中進度報告

探討

HPK1 相關的磷酸激活酶 (kinases) 對 T 淋巴細胞功能的影響及調控

(Function and Regulation of HPK1-Related Ste20 Kinases (HRKs) in T Cells)

計畫類別：X 個別型計畫 □ 整合型計畫

計畫編

號：

NSC 95-2320-B-006-028

執行期間： 95 年 2 月 1 日至 95 年 7 月 31 日

計畫主持人：

凌 斌

共同主持人：

計畫參與人員： 1

成果報告類型(依經費核定清單規定繳交)：X 精簡報告 □完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

□涉及專利或其他智慧財產權，□一年□二年後可公開查詢

Function and Regulation of HPK1-Related Ste20 Kinases (HRKs) in T Cells

ABSTRACT

T cells play an essential role in the regulation of immune responses. T-cell development, activation, differentiation and apoptosis are regulated by various intracellular signaling cascades. One of these signaling cascades is the mitogen-activated protein kinase (MAPK) pathway. Hematopoietic progenitor kinase 1 (HPK1) is a lymphoid-specific Ste20-related kinase and functions upstream of JNK, a member of MAPK family shown to involve in T-cell development and differentiation. Others and we first found that HPK1 is implicated in T- and B-cell antigen receptor signaling. A recent study demonstrated that an HPK1-related Ste20 kinase, called MINK (Misshapen/NIKs-related kinase), links the T-cell receptor (TCR) signal to the JNK pathway for thymocyte negative selection during T-cell development. Several other human Ste20-like kinases have a similar structure to HPK1. Here they are designated HPK1-related Ste20 kinases (HRKs), including GCK, GCKR/KHS, GLK, and HGK/NIK. The potential roles of these HRKs in T-cell signaling and functions remain unknown. We hypothesized that these HRKs are involved in T-cell signaling and play functional roles in T cells. Through this project, we have provided the following preliminary findings. First, results from amino acid sequence analysis showed that HPK1-related Ste20 kinases (HRKs), including GCKR/KHS, GLK, and HGK/NIK, contain the proline-rich motifs matching the consensus binding sites for interaction with SH2/SH3 adaptors (such as Grb2, Gads, Nck, and Crk). Secondly, we found that

GCKR/KHS and HGK, like HPK1, became tyrosine-phosphorylated after T-cell stimulation. Thirdly we aimed to determine which of SH2/SH3 adaptors might mediate the TCR signal to HPK1. Our preliminary results revealed that Nck mutants block HPK1 kinase induction after TCR stimulation. Together, data derived from this project highlight the potential involvement of other HPK1-related Ste20 kinases in T-cell signaling and function.

探討 HPK1 相關的磷酸激活酶 (kinases) 對 T 淋巴細胞功能的影響及調控

(Function and Regulation of HPK1-Related Ste20 Kinases (HRKs) in T Cells)

T 淋巴細胞 (T-lymphocytes) 在免疫系統扮演非常重要的功能角色 , 而 T 淋巴細胞的發育 (development)‚ 活化 (activation)‚ 分化 (differentiation) 及凋零 (apoptosis) 的機制是由細 胞內眾多訊息傳遞鏈 (signaling pathways) 所調控◦ Mitogen-activated protein kinase (MAPK) 訊息傳遞鏈便是其中之一◦ Hematopoietic progenitor kinase 1 (HPK1) 是存在 MAPK 訊息傳 遞的上游分子, 而且它只表現在免疫組織及細胞◦ 經由我們及其他實驗室的研究證明

HPK1 會參與 T 及 B 淋巴細胞 antigen receptor signaling ◦ 另一個 HPK1 相關的 磷酸激活酶 (kinase) MINK (Misshapen/NIKs-related kinase), 最近也被證明參與 T 淋巴細胞的早期發育◦ 基於上述發現, 我推測其他 HPK1 相關激活酶非常有可能參與 T 細胞的訊息傳遞, 並進一步 調控 T 淋巴細胞的功能◦ 在此研究計劃案我將測試四個與 HPK1 相關磷酸激活酶

(HPK1-related kinases), termed HRKs, 了解它們是否有可能參與 T 細胞內的訊息傳遞, 特 別是T-cell antigen receptor (TCR)及 CD28◦ 若發現是肯定,我們將進一步探討 HRKs 對 T 淋 巴細胞功能的調節及影響, 最後也將探討這些 HRKs 在 T 淋巴細胞中是如何被調控 ◦ 經 由此研究 , 我們期望能發現新的 HPK1-related kinases 參與調節 T 細胞功能, 更長遠影響 是經由調控HRKs 去改變 T 細胞參與的免疫反應, 進而能改善治療過敏,自體免疫疾病◦

BACKGROUND

Role of HPK1 in lymphocyte antigen receptor signaling

HPK1 is a hematopoietic-specific upstream activator of JNK (Jun N-terminal Kinase) and NF-κB transcription factors, suggesting a potential role for HPK1 in the regulation of immune responses 345. Previously we and other groups demonstrated that HPK1 is involved in T- and B-cell antigen receptor signaling. HPK1 becomes activation and tyrosine phosphorylated upon TCR or BCR stimulation 67. Lymphocyte-specific Src family kinases (Lck and Lyn) and Syk family kinases (Syk and Zap70) are involved in the regulation of HPK1 activation and tyrosine phosphorylation. Phosphorylation of Tyr381 on human HPK1 (or Tyr-379 on mouse HPK1) provides a binding site for interaction with the SH2 domains of SLP-76-related adaptors (SLP-76 and BLNK), leading to optimal HPK1 activation 58. In addition, several SH3 domain-containing adaptors (Grb2, Gads, Nck and Crk) interact with HPK1 and function as upstream regulators to link HPK1 to lymphocyte antigen receptors (TCR and BCR) and receptor tyrosine kinases (EGFR and PDGFR). These findings establish that HPK1 is the first GCK family member to involve in T and B lymphocyte antigen receptor signaling. Evidence from the molecular and biochemical analyses has established the involvement of HPK1 in lymphocyte antigen receptor signaling. However, more studies are needed to determine the functional role of HPK1 in lymphocytes and other immune cells.

MINK (Misshapen/NIKs-related kinase) is essential for thymocyte negative selection during T-cell development

The JNK pathway has been suggested to play a critical role in the T-cell negative selection in thymus 9. However the underlying mechanism of JNK-mediated T-cell negative selection is unclear yet. Using small interference RNA (siRNA)-based knockdown approach, McCarty et al. recently demonstrated that MINK, a member of human STE20-related kinases, is an essential signaling molecule linking the TCR signal to negative selection 10. They first found that

expression of MINK is selectively elevated in CD4+CD8+ double-positive (DP) thymoctye stage, suggesting a potential role for MINK in T-cell development. In MINK-knockdown mice they found that the single-positive thymocytes is significantly increased and the deletion of

HPK1-related Ste20 kinases (HRKs) and T-cell signaling

Although HPK1 and MINK have appeared as critical mediators in T-cell signaling , the roles of other HPK1-related Ste20 kinases (HRKs) in T cells are still unknown. We are interested in studying the potential roles of HRKs in T-cell signaling and function. Based on the studies of HPK1, we propose a hypothesis for HPK1 and HRKs as follow: T-cell surface receptors (TCR or CD28) => Tyrosine kinases => Adaptors (Grb2, Nck, and Crk) => HPK1 and HRKs => the MAPK pathway and/or other pathways (e.g., NF-κB) => T-cell responses (e.g., activation, differentiation, or apoptosis). HPK1 and HRK1 may play a redundant role in transmitting the TCR signal to the downstream pathways. Otherwise they have a unique role in T cells in response to different receptor signals.

RESULTS AND DISCUSSSION

HPK1-related Ste20 kinases (HRKs) contain the proline-rich motifs for interaction with SH2/SH3 adaptors.

Our and other groups’ studies have indicated that HPK1 is involved in T-cell receptor (TCR) signaling probably through several SH2/SH3 adaptors, including Grb2, Gads, Nck, and/or Crk 6,7,11. HPK1 differentially interacts with these adaptors via its four proline-rich motifs 1,2,12. Given these facts, we first attempted to determine whether other HPK1-related family kinases have similar proline-rich motifs for interaction with above-mentioned SH2/SH3 adaptors. By amino acid sequence analysis, we found that several HPK1-related family kinases contain the proline-rich motifs matching the Grb2 or Crk SH3 domain consensus binding site (Table 1). Using the UniGene's EST ProfileViewer

(http://www.ncbi.nlm.nih.gov/UniGene), we also examined the expression profiles of these HPK1-related family kinases. We found that several of them are expressed in the lymphoid tissues (such as spleen, lymph nodes, and bone marrow) (Table 1). These results suggest that these HPK1-related family kinase are potentially implicated in lymphocyte signaling.

Kinase Number of Proline-rich motif (-P-x-x-P-) Lymphoid tissue (Spleen, LN) HPK1 4 (Grb2, Crk, and Nck) + MINK 2 (Nck or Grb2) + KHS/GCKR 2 (Grb2 or Crk) + HGK/NIK 2 (Nck) + GLK 1 (Grb2 or Crk) +

_{Table1. Analysis of the proline-rich motifs of the HPK1-related family kinases.}

All sequences are obtained from the NCBI database and analyzed for the proline-rich motifs as described previously 1,2. The parenthesis indicates the known or possible adaptors for interactions with each kinase. Lymphoid tissue expression is obtained by analyzing the UniGene's EST Profile.

first attempted to establish the T-cell activation by TCR/CD3 or PV treatment in the lab. Results from anti-phosphotyrosine immunoblot showed that tyrosine-phosphorylated molecules in Jurkat lysates are increased after 5 min of TCR/CD3 stimulation and PV treatment (Fig. 1A). Furthermore, we performed the immunoprecipitation-immunoblot analysis to determine whether HRKs become tyrosine-phosphorylated after PV treatment. Our preliminary results showed that KHS/GCKR and HGK, like HPK1, become

tyrosine-phosphorylated in PV-treated Jurkat T cells (Fig. 1B). These results suggested that in addition to HPK1, other HRKs play a role in T-cell activation.

WB: Anti-pTyr

1 2 3 4

Un tre ate d ( 50 µg ) Un tre ate d ( 5 µ g) CD 3-t rea ted (5 0 µ g) PV -tre ate d ( 5 µ g) WB: Anti-pTyr

1 2 3 4

Un tre ate d ( 50 µg ) Un tre ate d ( 5 µ g) CD 3-t rea ted (5 0 µ g) PV -tre ate d ( 5 µ g)

1A

1B

IP: anti-HPK1 IP: anti-KHS IP: anti-HGK WB: anti-pTyr WB: anti-pTyr WB: anti-pTyr WB: anti-HPK1 WB: anti-KHS WB: anti-HGK PV − IP: anti-HPK1 IP: anti-KHS IP: anti-HGK WB: anti-pTyr WB: anti-pTyr WB: anti-pTyr WB: anti-HPK1 WB: anti-KHS WB: anti-HGK PV −

Fig. 1. Tyrosine phosphorylation of HPK1, KHS, and HGK in activated Jurkat T cells. 7

(A) Jurkat T cells (5 × 10 ) were left untreated or treated with an anti-CD3 mAb (10 µg/ml) or pervanadate (50 µM) for 5 min. Untreated and treated cells were lysed in RIPA buffer and ind amount of lysates are subjected to the immunoblot analysis with an anti-phosphotyrosine mAb (4G10). (B) Untreated or pervanadate-treated Jurkat T cell lysates (5 × 10

icated

K

7_{) were subjected to}

immunoprecipitation with an anti-HPK1 antibody (N-19), anti-KHS antibody (N-19) or anti-HG antibody (M-190). Immunoprecipitation samples were then analyzed by immunoblotting with an anti-phosphotyrosine mAb (4G10), and subsequently reprobed by an anti-HPK1 antibody (N-19) anti-KHS antibody (N-19) or anti-HGK antibody (M-190). Data are representative of three independent experiments.

ck may mediate the TCR signal to HPK1

An important issue in studying HPK1 signaling in T cells is to determine what Sh2/SH3

n s

).

is

N

adaptors may mediate the TCR signal to HPK1 activation. It is likely that other HRKs employ the similar mechanistic mechanism linking TCR. Recently Nck adaptor has bee shown to play an important role in TCR signaling to downstream events 1013. We were thu interested in exploring whether Nck is implicated in TCR signaling to HPK1. To this end, we first found that the third SH3 domain of Nck is responsible for HPK1 interaction (Fig. 2A Moreover, TCR/CD3-indcued HPK1 kinase induction could be blocked by a Nck mutant containing a mutation on the third SH3 domain (Fig. 2B). These data suggested that Nck critical for HPK1 activation during TCR stimulation.

Direct WB: αHA Fla g-HPK 1 + Ha-N ck-α WB: αHPK1 1 2 3 4 5 6 WB:anti-HA Flag -HPK 1 + H a-Nc k-α W38K Flag -HPK 1 + Ha-N ck-α W14 3K Flag -HPK 1 + Ha-N ck-α W22 9K Flag -HPK 1 + Ha-N ck-α W38 /143 /229 K Flag -HPK 1 IP:anti-HPK Direct WB: αHA Fla g-HPK 1 + Ha-N ck-α WB: αHPK1 1 2 3 4 5 6 WB:anti-HA Flag -HPK 1 + H a-Nc k-α W38K Flag -HPK 1 + Ha-N ck-α W14 3K Flag -HPK 1 + Ha-N ck-α W22 9K Flag -HPK 1 + Ha-N ck-α W38 /143 /229 K Flag -HPK 1 IP:anti-HPK

2A

5’ αCD3

H

PK

1

+ V

ec

to

r

HP

K

1

+

V

ec

to

r

H

PK

1

+

N

ck

α

H

PK

1

+

N

ck

α−

W

23

5K

H

PK

1

+

N

ck

α−

R

31

2K

−

+

+

+

+

1

2

3

4

5

MBP

5’ αCD3

H

PK

1

+ V

ec

to

r

HP

K

1

+

V

ec

to

r

H

PK

1

+

N

ck

α

H

PK

1

+

N

ck

α−

W

23

5K

H

PK

1

+

N

ck

α−

R

31

2K

−

+

+

+

+

1

2

3

4

5

MBP

2B

i

F g. 2. Nck adaptor mediates the TCR signal to HPK1 activation in Jurkat T cells.

ck

(B)

Ab (A) HEK293T cells were transfected with HPK1 alone or in combination with various N

constructs. Anti-HPK1 immunoprecipitates were analyzed by immunoblotting with an anti-HA mAb (12CA5), and subsequently reprobed by an anti-HPK1 antibody (N-19). Jurkat T cell lysates (2 × 107_{) were transfected with HPK1 alone or in combination with}

various Nck constructs. Transfected cells were left untreated or stimulated by anti-CD3 m (OKT3) for 5 min. Then immunocomplex kinase assays were performed to examine HPK1 kinase activity.

. Ling, P. et al. Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crk

2. he germinal center kinase (GCK)-related protein kinases HPK1 and ins.

3. eyer, C.F. & Tan, T.H. Human HPK1, a novel human

4. , a hematopoietic protein kinase activating the SAPK/JNK pathway.

5. ontaining src homology 2 domain (BASH) links B cell

94,

6. K1 is activated by lymphocyte antigen receptors and negatively regulates

7. oietic progenitor kinase 1 in T cell receptor

8. e 1 associates physically and functionally

9. K pathway regulates the In vivo deletion of immature

10. the negative selection of

11. nteracts with hematopoietic

12. r proteins can link tyrosine kinases to a Ste20-related

13. Alarcon, B. Recruitment of 1

and CrkL leads to synergistic activation of c-Jun N-terminal kinase. Mol Cell Biol 19, 1359-68 (1999).

Oehrl, W. et al. T

KHS are candidates for highly selective signal transducers of Crk family adapter prote

Oncogene 17, 1893-901 (1998).

Hu, M.C., Qiu, W.R., Wang, X., M

hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade. Genes Dev

10, 2251-64 (1996).

Kiefer, F. et al. HPK1

Embo J 15, 7013-25 (1996).

Tsuji, S. et al. B cell adaptor c

receptor signaling to the activation of hematopoietic progenitor kinase 1. J Exp Med 1 529-39 (2001).

Liou, J. et al. HP

AP-1. Immunity 12, 399-408 (2000). Ling, P. et al. Involvement of hematop

signaling. J Biol Chem 276, 18908-14 (2001). Sauer, K. et al. Hematopoietic progenitor kinas

with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes. J Biol Chem

276, 45207-16 (2001).

Rincon, M. et al. The JN

CD4(+)CD8(+) thymocytes. J Exp Med 188, 1817-30 (1998). McCarty, N. et al. Signaling by the kinase MINK is essential in autoreactive thymocytes. Nat Immunol 6, 65-72 (2005).

Ma, W. et al. Leukocyte-specific adaptor protein Grap2 i

progenitor kinase 1 (HPK1) to activate JNK signaling pathway in T lymphocytes.

Oncogene 20, 1703-14 (2001).

Anafi, M. et al. SH2/SH3 adapto

protein kinase, HPK1. J Biol Chem 272, 27804-11 (1997). Gil, D., Schamel, W.W., Montoya, M., Sanchez-Madrid, F. &