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.

Adapter proteins function by mediating the rapid and speci®c assembly of multi-protein complexes during the signal transduction which guards proliferation, dierentiation and many functions of higher eukaryotic cells. To understand their functional roles in dierent cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the ®rst SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the ®rst SH3 domain of CRKL is direct and occurs via proline-rich motifs in the Cterminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by coimmunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.

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Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.

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