We have investigated the potential interaction of HPK1 with several SH2/SH3 adapter proteins, including Grb2, Nck, Crk, and CrkL. Here we provide evidence from binding assays and kinase assays to demonstrate the functional interaction of HPK1 with Crk and CrkL. We showed that fusion proteins GST-Crk and GST-CrkL pulled down HPK1 from Jurkat T-cell lysates and that HPK1 formed complexes with Crk and CrkL in mammalian cells. Also, the Crk proteins enhanced the recruitment of HPK1 to the EGF receptor, supporting the previous report that SH2/SH3 adapter proteins link surface receptors to HPK1 (1
). Although the expression of HPK1 is mainly restricted to hematopoietic cells, SH2/SH3 adapter proteins such as Grb2, Crk, and CrkL are ubiquitously expressed in a wide range of tissues and cells, including T cells, B cells, hematopoietic progenitor cells, and other cells (25
). Based on the previous evidence and our own data, we speculate that HPK1 may form complexes with Crk and CrkL in hematopoietic cells constitutively or upon extracellular stimulation. The proline-rich motifs of HPK1 mediated the complex formation between HPK1 and various SH2/SH3 adapter proteins (Fig. ). Particularly, the second proline-rich motif containing the Crk N-SH3-binding consensus sequence (-P-x-L-P-x-K-) was critical for the binding of HPK1 to Crk and CrkL, whereas the first and second proline-rich motifs containing the Grb2 N-SH3-binding consensus sequence (-P-x-x-P-x-R/K-) were implicated in the binding of HPK1 to Grb2. The third proline-rich motif did not play a role in the interaction of HPK1 with the SH2/SH3 adapter proteins tested; however, it may be involved in the association of HPK1 with other SH3 domain-containing proteins. Since the fourth proline-rich motif is not conserved between human and mouse HPK1, it may not play a critical functional role for HPK1. Consistent with previous findings (18
), our data showed that lysine-400 in the HPK1 second proline-rich motif is critical for the recognition of Crk and CrkL by HPK1 (Fig. C).
SH2/SH3 adapter proteins, which themselves have no enzymatic activity, are signaling modulators to form multiple protein complexes selectively and thereby to transmit upstream signals to downstream targets. SH2/SH3 adapter proteins usually localize close to the cell membrane, and some are even directly associated with surface receptors or the cytoskeleton (24
). For instance, Grb2 and Crk are recruited to activated EGF receptors via their SH2 domains (2
). Crk is also recruited indirectly to the nerve growth factor receptor through Shc (23
) and to integrin through Paxillin, a constituent of focal adhesions (5
). Thus, SH2/SH3 adapter proteins play their roles in the very upstream or initial events during signal transduction. According to the features of adapter proteins and our data, it is likely that the differential interaction of HPK1 with various SH2/SH3 adapter proteins provides a mechanism for HPK1 to integrate different upstream signals to the JNK signaling cascade. A similar phenomenon may occur in other mammalian Ste20-related MAPKKKKs containing the proline-rich motifs, such as NIK, GLK, and KHS. It will be of interest to dissect the HPK1-mediated JNK signaling pathways by using specific HPK1 proline-rich motif mutants and the related adapter mutants. This approach will be useful in characterizing parallel JNK signaling cascades mediated by other MAPKKKKs.
Through kinase assays, we have shown that the Crk proteins directly activated HPK1 and also cooperated with HPK1 to activate JNK synergistically (Fig. ). These results indicated that the Crk proteins not only physically interacted with HPK1 but also functionally regulated HPK1-mediated JNK activation. A previous study demonstrated that C3G, a Crk-interacting protein, mediates the downstream signaling of v-Crk-induced JNK activation (53
). Like C3G, HPK1 bound Crk via recognition of the Crk N-SH3 domain (Fig. B), suggesting that HPK1 and C3G may be at the same level of the parallel Crk-mediated JNK signaling pathways. We further demonstrated that expression of HPK1-PR, the region responsible for binding to Crk and CrkL, blocked JNK activation by Crk and CrkL and that the downstream effectors of HPK1, such as MEKK1, TAK1, and SEK1, were also involved in Crk-induced JNK activation (Fig. ). Although it is possible that the overexpression of HPK1-PR may compete with endogenous C3G for Crk binding, the evidence from the kinase assays (Fig. and ) strongly suggests that HPK1 is one of the Crk downstream effectors for the Crk-mediated signaling pathways. Therefore, we propose the model for the HPK1-mediated JNK signaling cascade shown in Fig. . HPK1 receives the upstream signals through its differential interaction with various SH2/SH3 adapter proteins and subsequently transmits these signals to downstream targets (MEKK1 and TAK1) via its kinase domain or distal regulatory region. Afterwards, the signals are transmitted to a target further downstream (SEK1), thereby leading to JNK activation. Although HPK1 was shown to associate EGF receptor in transfected COS-1 cells, the surface receptors, which recruit the Crk-HPK1 and CrkL-HPK1 complexes in hematopoietic cells, are yet to be determined. By identifying the surface receptors that activate HPK1, we may elucidate the entire HPK1-mediated JNK signaling cascade from the cell membrane to the nucleus.
FIG. 9 The proposed model for the HPK1-mediated JNK signaling cascade. The adapter proteins Crk and CrkL couple surface receptors to HPK1. Subsequently, HPK1 transmits the upstream signals to downstream MAPKKKs, such as MEKK1 and TAK1, which transmit the signals (more ...)
Optimal IL-2 induction, a central event of T-cell activation, requires two major signals triggered by costimulation of the T-cell receptor (TCR) and CD28 or mimicked by incubating T cells with phorbol ester (i.e., PMA) and Ca2+
). Early studies have indicated that AP1 binds the IL-2 promoter (31
) and forms a complex with NF-AT to induce IL-2 activation (33
). Another study also showed that JNK is involved in the integration of the costimulatory signals CD3 plus CD28 or PMA plus Ca2+
ionophore in T cells (49
). However, the response of JNK to Ca2+
ionophore is T cell specific, suggesting that a cell type-specific component plays a role in this event (49
). As a hematopoietic upstream activator of JNK and AP1, HPK1 may be involved in IL-2 induction in T cells. By CAT reporter assays, we found that the HPK1 mutants, HPK1-KD(M46) and HPK1-PR, blocked IL-2 activation by T-cell stimuli (PHA, PMA, and ionomycin) in Jurkat T cells (Fig. ). Inhibition of IL-2 induction by HPK1-PR may be due to the interruption of upstream signals mediated by the SH2/SH3 adapter proteins. The SH2/SH3 adapter proteins, Crk and Grb2, are known to form distinct signaling complexes during TCR stimulation (3
), suggesting that these adapter proteins are involved in the TCR signaling pathway. Grb2 is also shown to couple upstream tyrosine kinases such as v-Src to HPK1 (1
). In addition, we also found that Crk-R38K, a Crk SH2 domain mutant, blocked IL-2 induction. These observations further support the idea that HPK1-PR may block the transmission of upstream signals to the IL-2 promoter. The mechanism by which HPK1-KD(M46) inhibits of IL-2 induction is unclear. One possible explanation is that HPK1-KD(M46) may compete with wild-type HPK1 for the binding of the downstream targets (MAPKKKs), which are essential for JNK and AP1 activation. More studies are needed to define the role of HPK1 in IL-2 activation. Future studies will examine whether T-cell costimulatory signals, TCR plus CD28 or PMA plus Ca2+
ionophore, can activate HPK1.
The phosphorylation of Crk and CrkL by HPK1 further supported the notion that HPK1 bound Crk and CrkL. Interestingly, our data from phosphoamino acid analysis revealed that Crk and CrkL were phosphorylated mainly on serine and threonine (Fig. B). We noticed that phosphorylated Crk and CrkL also contained some phosphotyrosine residues. Since HPK1 is a serine-threonine kinase, HPK1 is unlikely responsible for this tyrosine phosphorylation. The c-Abl tyrosine kinase was previously shown to bind the HPK1 proline-rich motifs via its SH3 domain (17
). Thus, these tyrosine phosphorylations are most likely due to the coimmunoprecipitation of c-Abl with HPK1. Previous evidence indicated that c-Abl binds the Crk N-SH3 domain and phosphorylates Crk on tyrosine 221 (Y221). This phosphorylated Y221 then provides a binding site for the Crk SH2 domain to form an intramolecular interaction resulting in an uncomplexed Crk molecule (12
). Similarly, tyrosine residue 207 (Y207) in CrkL has been identified to function as a negative regulatory site (45
). Although HPK1 is unlikely to phosphorylate Crk and CrkL on Y221 and Y207, serine and threonine phosphorylation of the Crk proteins by HPK1 might still play a role in the feedback regulation of the Crk proteins. Future studies will focus on determining the phosphorylation sites of Crk and CrkL induced by HPK1 and the functional significance of these phosphorylations. These studies will provide critical insights for understanding the Crk- and CrkL-mediated cellular responses. It will be also interesting to investigate whether HPK1, like c-Abl and C3G, plays a role in cell transformation induced by the Crk proteins.