Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS.
MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.
Resing
K A
KA
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309.
Mansour
S J
SJ
Hermann
A S
AS
Johnson
R S
RS
Candia
J M
JM
Fukasawa
K
K
Vande Woude
G F
GF
Ahn
N G
NG
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D017232:Oncogene Proteins v-mos / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017232:Oncogene Proteins v-mos; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases