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. 1998 Nov 1;12(21):3369–3381. doi: 10.1101/gad.12.21.3369

Figure 2.

Figure 2

JNKK1 is the preferred MAPKK substrate for MEKK1. (A) MEKK1 preferentially activates JNKK1 in mammalian cells. Cos-1 cells were transiently transfected with increasing amounts of ΔMEKK1, together with HA-tagged MAPKKs. Immunoblot analyses and immunecomplex kinase assays were performed as described in Fig. 1, using recombinant, catalytically inactive MAPKs as substrates. Fold stimulations of MAPKK activities were calculated as in Fig. 1 and are indicated below each autoradiograph. As references, JNKK1, MKK3, and MKK6 activities were stimulated by UV irradiation of transfected cells, and MEK1/2 by TPA treatment. (B) JNKK1 is the preferred MAPKK substrate for MEKK1 in vitro. Kinase assays (see Materials and Methods) were performed using purified recombinant GST–ΔMEKK1 as the kinase and purified recombinant GST–MAPKK fusion proteins, as well as JNKK1 truncation mutants—JNKK1(78–399) or JNKK1(89–399)—as substrates. Incorporation of 32P was determined by scintillation counting. Data were corrected for MAPKK autophosphorylation and fit to the Michaelis–Menten equation. (Inset) The substrate specificity constants (Vmax/Km).