The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins

Abstract

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.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways

“…RIPA-lysates were prepared as described (Oehrl et al, 1998). The antibodies used for immuno-complex kinase assay were selected for best speci®city: c-Abl was precipitated from NIH3T3 cell lysates with anti-Abl (Ab-3), Arg with polyclonal anti-Arg serum from Abl 7/7 /cell lysates, Bcr-Abl with anti-Bcr from NIH3T3 Bcr-Abl cell lysates and Tel-Abl from Ba/F3 Tel-Abl cell lysates with anti-Myc.…”

“…For analysis of CRKL/Tel-Abl complexes, Ba/F3 Tel-Abl cells and parental Ba/F3 cells were extracted with 1% Triton X-100 lysis bu er as described previously (Oehrl et al, 1998). After preclearing on protein A-Sepharose and preabsorption on immobilized immunoglobulins from normal rabbit serum, the supernatant was ®nally incubated overnight with immobilized anti-CRKL or an equal amount of immunoglobulins from non-immunized rabbits as indicated.…”

Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1)

“…JNK cascade (Hu et al, 1996;Wang et al, 1997;Zhou et al, 1999). HPK1 may mediate extracellular signals through interaction with Src-homology 3 (SH3) domaincontaining adaptor molecules (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Here we report that HPK1 was cleaved by caspase activity during apoptosis.…”

“…One interesting structural feature of HPK1 is its four proline-rich motifs (putative SH3 domain-binding sites) in its regulatory region (Hu et al, 1996). We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via these proline-rich motifs (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Co-expression of Crk with CrkL enhances HPK1 activity through mechanisms yet unknown (Ling et al, 1999).…”

“…We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via proline-rich motifs in the C-terminal regulatory region (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Cleavage of HPK1 by caspases separates the Nterminus of HPK1 (containing kinase domain and the ®rst proline-rich motif) from the C-terminus (containing the remaining three proline-rich motifs (Figure 2a).…”