MTK1 N-terminal region inhibits its kinase activity. (A) Yeast strain FP75 (Δssk2 Δssk22 Δste11) was transformed with either a centromeric vector plasmid (p414ADH) or one of its derivatives containing various MTK1 segments. Transformed cells were spread on YPD agar plates supplemented with (+) or without (−) 1.5 M sorbitol. FP75 cells grow on plates containing sorbitol only when the MTK1 kinase activity is expressed. The segment of MTK1 cDNA sequence included in each construct is indicated, both schematically and numerically, in panel B. (B) The topmost bar represents the full-length MTK1 cDNA (amino acids from positions 22 through the C terminus [position 1607]), and other bars represent deletion derivatives. The gray section indicates the GADD45-binding site, whereas the filled section indicates the kinase catalytic domain. Funct. compl., functional complementation; +, complementation; −, no complementation.

Activation of full-length MTK1 (MTK1-FL) by GADD45 proteins. Yeast strain FP75 was transformed with two expression plasmids (p414TEF for MTK1-FL and p426GAL for GADD45) with (+) or without (−) the indicated cDNA sequences. p426GAL is a multicopy plasmid with the galactose-inducible GAL1 promoter. Transformed cells were spread on YPGal agar plates supplemented with (+) or without (−) 1.5 M sorbitol.

Two-hybrid interaction analysis between MTK1 and MKK6. (A) Interaction of various MTK1 segments fused to the GAL4 activation domain (ACT-MTK1 constructs) with MKK6-K/A fused to the LexA DB (DB-MKK6-K/A) was tested. Vectors used in these experiments were pACTII and pBTM118. Segments of MTK1 included in the ACT-MTK1 constructs are indicated. A minus sign in the ACT-MTK1 column indicates the vector without an insert. Yeast L40 cells were transformed with two plasmids (ACT-MTK1 and DB-MKK6-K/A constructs) as indicated. −, vector without an insert; +, vector with an insert. Transformed L40 cells were spread on the appropriate synthetic agar plates supplemented with (+His) or without (−His) histidine. A kinase-defective MKK6-K/A mutant was used to reduce the toxic effect of MTK1 expression. MTK1(1309-1607) encodes the kinase catalytic domain, whereas MTK1(22-1341) encodes the N-terminal noncatalytic domain. In situ β-Gal assays gave essentially identical results (not shown). (B) Summary of the experiments in panel A and additional two-hybrid tests. The MTK1 coding sequence is represented by the horizontal bar. Gray and filled boxes are the GADD45-binding domain and the kinase catalytic domain, respectively. The segment between amino acid positions 253 and 553 is required for autoinhibition of the MTK1-MKK6 interaction. +, growth on −His plates; −, no growth on −His plates. (C) Summary of two-hybrid analyses for the MTK1 binding site in MKK6. Binding of full-length (positions 1 to 334) and various deletion constructs of DB-MKK6-K/A was tested with ACT-MTK1(1309-1607) as a bait. The MKK6 coding sequence is represented by the horizontal bar. Filled boxes represent the kinase catalytic domain. The C-terminal noncatalytic region of MKK6 is necessary to interact with the MTK1 kinase domain. +, growth on −His plates; −, no growth on −His plates.

Three-hybrid interaction analysis. Yeast L40 cells were transformed with three plasmids (ACT-MTK1, DB-MKK6-K/A, and GADD45 constructs) as indicated. A minus sign indicates the corresponding vector (pACTII, pBTM118, or pRS422-THL) without an insert; a plus sign indicates the vector with an insert. Transformed cells were spread on the appropriate synthetic agar plates supplemented with (+His) or without (−His) histidine.

Mutants of full-length MTK1 that can interact with MKK6 in the absence of GADD45. Yeast L40 cells were transformed with an ACT-MTK1 construct (pACT-MTK1-FL wild type or one of its derivatives, as indicated on the figure) and a common DB-MKK6-K/A construct (pBTM-MKK6-K/A). Transformed yeast cells were spread on the appropriate synthetic agar plates supplemented with (+His) or without (−His) histidine. Mutations are designated by a one-letter amino acid code: L534Q, for example, stands for a mutation of leucine at the 534th amino acid position to glutamine. ACT-MTK1(1309-1607), which contains only the MTK1 kinase catalytic domain, was used as a positive control.

Locations of the constitutively active MTK1 mutations. (A) The positions of the 11 MTK1 mutations are shown along a bar representing the MTK1 coding sequence. Four regions (A1, A2, B1, and B2) are indicated. The GADD45-binding domain and the kinase catalytic domain are also shown. (B) The predicted three-dimensional locations of the two mutations in region B2 (I1360M and M1414I) are shown by using the crystallographic structure of the PAK1 kinase domain (19).

MTK1 mutants that can interact with MKK6 in the absence of GADD45 are constitutively active in yeast cells. The osmosensitive yeast strain FP75 was transformed with p414ADH-MTK1-FL or one of its derivatives, as indicated in the cDNA sequences. K/R is the catalytically inactive K1371R mutation in the MTK1 kinase domain and was used as a negative control. MTK1(1051-1607) is a constitutively active N-terminal truncation mutant used here as a positive control. Transformed cells were spread on YPD agar plates supplemented with (+) or without (−) 1.5 M sorbitol.

Kinase activities of MTK1 mutants. (A and B) In vitro kinase activities of MTK1 mutants. HeLa (A) or Cos-7 (B) cells were transiently transfected with Flag-tagged MTK1 (wild type or mutant) with or without HA-tagged GADD45γ. Flag-tagged MTK1 was immunoprecipitated from cell extracts, and its kinase activity was assayed in vitro with the substrate GST-MKK6-K/A in the presence of [γ-³²P]ATP. Phosphorylated GST-MKK6-K/A was detected by autoradiography following SDS-polyacrylamide gel electrophoresis. Expression levels of Flag-MTK1 and HA-tagged GADD45γ were monitored by immunoblotting. In panel A, phosphorylated GST-MKK6-K/A was also detected by PhosphorImager (Molecular Dynamics) and quantitated. Kinase-dead MTK1-K/R was used as a negative control, and N-terminally truncated MTK1(1322-1607) was used as a positive control. The expression level of Flag-tagged MTK1(1322-1607) was comparable to that of the others, although it is not visible in this figure due to its smaller size. (C) In vivo kinase activities of MTK1 mutants. Cos-7 cells were transiently transfected with Flag-tagged MTK1 (wild type or mutant) together with HA-tagged MKK6-K/A. The phosphorylation status of transfected HA-MKK6-K/A was detected by immunoblotting with phospho-MKK6-specific antibody. Expression levels of HA-MKK6-K/A and Flag-MTK1 were also monitored by immunoblotting. wt, wild-type.

Two-hybrid interaction analysis between N- and C-terminal MTK1 segments. (A) Sets of ACT-MTK1 N-terminal constructs and DB-MTK1 C-terminal constructs, as shown schematically by horizontal bars and numerically in the table, were transformed into L40 tester cells. The presence and absence of the N-C interaction, as deduced from growth on histidine-deficient plates and β-Gal expression, are indicated by plus and minus signs, respectively. The GADD45 binding site (gray), kinase catalytic domain (black), and four regions (A1, A2, B1, and B2) are also indicated. (B) Some, but not all, constitutively active MTK1 mutants are defective in the N-C interaction, as detected by two-hybrid analysis. The ACT-MTK1-N construct contains the MTK1(22-1103) segment, and the DB-MTK1-C construct contains the MTK1(1104-1607) segment. Either a wild-type (wt) or a mutant sequence, as indicated in the figure, was tested in each pair. A minus sign indicates the corresponding vector (pACTII or pBTM118) without an insert. +His, with histidine; −His, without histidine.

A speculative model of MTK1 autoinhibition by its N-terminal region and activation by GADD45 binding. (A) Schematic representation of MTK1 structure. The GADD45 binding site, kinase inhibitory domain, and kinase catalytic domain are shown. Also shown are the locations of four regions (A1, A2, B1, and B2) defined by constitutively active MTK1 mutations. (B) Model of MTK1 activation mechanism. In the autoinhibited conformation, the N-terminal inhibitory domain interacts with the C-terminal kinase domain so that substrates (such as MKK6) cannot interact. It is likely that there are multiple interactions between the N- and C-terminal regions. The A1 and B1 regions may interact strongly and serve as a clasp. Weaker interactions involving the kinase domain itself must also exist. Binding of GADD45 to a site near the inhibitory domain sterically interferes with the N-C interaction, making the kinase domain accessible to its substrates.