The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin ␤1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin ␤1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin ␤1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin ␤1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.Despite a number of recent reports on translocation of receptor tyrosine kinases (RTKs) to the nucleus (reviewed in references 8 and 52), the mechanism of how RTKs travel from the cell surface to the nucleus is still unknown. Transmembrane receptors serve as sensors, recognizing the growth factor in the extracellular environment and conveying the message through the signaling cascade to the nucleus. However, some RTKs, like the epidermal growth factor receptor (EGFR) family members (27,29,32,36,37,49,54), fibroblast growth factor receptor 1 (FGFR1) and FGFR3 and their splice variants (23,31,40,43,44,45,57), insulin receptor (41), and vascular endothelium growth factor receptor Flk1/KDR (13,21,34), are known to migrate to the nucleus either intact or as a fragment of proteolytic cleavage, interestingly, with or without the corresponding ligand. These nuclear RTKs have been shown to act as transcription factors (27,36,49,54) for genes like Cyclin D1 (27), FGF2 (38), and COX-2 (49) and modulators for induction of c-jun and cyclin D1 (40). Additionally, nuclear matrix binding of FGFR1 and insulin receptor (41, 45) has been shown to strategically position the receptors for involvement in the regulation of gene expression. More recently, the nuclear EGFR was shown to interact with well-known DNA binding transcriptional factors, such as STAT3 and E2F1, to regulate the expression of inducible nitric oxide synthase and B-Myb (17, 28). Overall, these reports support direct roles for RTKs in the nucleus, which represent a new class of RTK functions.However, the mechanism of their nuclear import is virtually unknown. We repor...

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Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.

Enterovirus 71 (EV71) causes fatal encephalitis in young children. However, there is no effective antiviral drug available for infected patients. Ribavirin is currently used for the treatment of several RNA virus infections clinically, so its anti-EV71 efficacy was evaluated. In vitro results showed that ribavirin effectively reduced the viral yields (with an IC50 of 65 microg/mL) and virus-induced cytopathic effect in human and mouse cell lines. In vivo results showed that ribavirin reduced the mortality, morbidity, and subsequent paralysis sequelae in infected mice by decreasing viral loads in tissues. Thus, ribavirin could be a potential anti-EV71 drug.

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Immune cell-speci®c adaptor proteins create various combinations of multiprotein complexes and integrate signals from cell surface receptors to the nucleus, modulating the speci®city and selectivity of intracellular signal transduction. Grap2 is a newly identi®ed adaptor protein speci®cally expressed in lymphoid tissues. This protein shares 40 ± 50% sequence homology in the SH3 and the SH2 domain with Grb2 and Grap. However, the Grap2 protein has a unique 120-amino acid glutamineand proline-rich domain between the SH2 and Cterminal SH3 domains. The expression of Grap2 is highly restricted to lymphoid organs and T lymphocytes. In order to understand the role of this speci®c adaptor protein in immune cell signaling and activation, we searched for the Grap2 interacting protein in T lymphocytes. We found that Grap2 interacted with the hematopoietic progenitor kinase 1 (HPK1) in vitro and in Jurkat T cells. The interaction was mediated by the carboxyl-terminal SH3 domain of Grap2 with the second proline-rich motif of HPK1. Coexpression of Grap2 with HPK1 not only increased the kinase activity of HPK1 in the cell, but also had an additive e ect on HPK1 mediated JNK activation. Furthermore, over expression of Grap2 and HPK1 induced signi®cant transcriptional activation of c-Jun in the JNK signaling pathway and IL-2 gene reporter activity in stimulated Jurkat T cells. Therefore, our data suggest that the hematopoietic speci®c proteins Grap2 and HPK1 form a signaling complex to mediate the c-Jun NH 2 -terminal kinase (JNK) signaling pathway in T cells. Oncogene (2001) 20, 1703 ± 1714.

Dengue is one of the most important vector-borne viral diseases. With climate change and the convenience of travel, dengue is spreading beyond its usual tropical and subtropical boundaries. Infection with dengue virus (DENV) causes diseases ranging widely in severity, from self-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical manifestations associated with severe DENV infection, yet the mechanisms remain unclear. Besides the direct effects of the virus, immunopathogenesis is also involved in the development of dengue disease. Antibody-dependent enhancement increases the efficiency of virus infection and may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cells and overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules lead to their abnormal activation or dysfunction. Molecular mimicry between DENV proteins and host proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development. For the development of a safe and effective dengue vaccine, the immunopathogenic complications of dengue disease need to be considered.

Herpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. Both primary and recurrent infections can induce diseases, such as encephalitis. In humans, the majority of encephalitis cases occur as a recurrent infection. However, in the past, numerous mouse studies documented that viral reactivation occurs efficiently in the ganglion, but extremely rarely in the brain, when assessed ex vivo by cultivating minced tissue explants. Here, we compare the brains and the trigeminal ganglia of mice latently infected with HSV-1 (strain 294.1 or McKrae) for levels of viral genomes and in vivo reactivation. The numbers of copies of 294.1 and McKrae genomes in the brain stem were significantly greater than those in the trigeminal ganglion. Most importantly, 294.1 and McKrae reactivation was detected in the brain stems earlier than in the trigeminal ganglia of mice treated with hyperthermia to reactivate latent virus in vivo. In addition, the brain stem yielded reactivated virus at a high frequency compared with the trigeminal ganglion, especially in mice latently infected with 294.1 after hyperthermia treatment. These results provide evidence that recurrent brain infection can be induced by the reactivation of latent virus in the brain in situ. IMPORTANCEHerpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. In the past, studies of viral reactivation focused on the ganglion, because efficient viral reactivation was detected in the ganglion but not in the brain when assessed ex vivo by cultivating mouse tissue explants. In this study, we report that the brain contains more viral genomes than the trigeminal ganglion in latently infected mice. Notably, the brain yields reactivated virus early and efficiently compared with the trigeminal ganglion after mice are stimulated to reactivate latent virus. Our findings raise the potential importance of HSV-1 latent infection and reactivation in the brain.