Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop.
Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.
Durkin
John T
JT
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, USA. jdurkin@cephalon.com
Holskin
Beverly P
BP
Kopec
Karla K
KK
Reed
Matt S
MS
Spais
Chrysanthe M
CM
Steffy
Brian M
BM
Gessner
George
G
Angeles
Thelma S
TS
Pohl
Jan
J
Ator
Mark A
MA
Meyer
Sheryl L
SL
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000378:metabolism; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D017346:Protein Serine-Threonine Kinases; D013912:Threonine; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C433243:MAP3K9 protein, human