Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

R Seger,D Seger,F.J Lozeman,N.G Ahn,L.M Graves,J.S Campbell,L Ericsson,M Harrylock,A.M Jensen,E.G Krebs

doi:10.1016/s0021-9258(18)35648-5

R Seger, D Seger + Show 8 more

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https://doi.org/10.1016/s0021-9258(18)35648-5

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Abstract

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.

Highlights

Protein Kinase Kinases Are Related to Yeast Signal from mature Xenopus oocytes [7] andepidermal growth factor (EGF)-stimulated A431 cells [8].While a single protein of 45 kDa structurally related to the yeast protein STE7 was isolated from Xenopus oocytes [9], two forms of the mitogen-activated protein kinase kinase (MAPKK)
A 46-kDa MAPKK purified from EGF-stimulated A431 cells [8]and a 45-kDa MAPKK purified from rabbit skeletal muscle2were usedto obtain protein sequence
Contained the sequences of 10 of the peptides obtained by Further proof that the cDNA clone encodes the correct protein sequencing plus all of the conserved kinase domains, MAPKK protein is providedby the overexpression of the strongly suggests that the two forms of the enzyme exist in translation product of MKKla in COS cellsI.n order to ensure the cell, possibly representing the 46- and 45-kDa forms optimal expression, analanine codon(GAC)was inserted described earlier in A431 cells [8].The absolute identity in after the startinmgethionine of the MKKlacDNA to provide nucleotide sequence in the common region of the molecules a guanosine in position +4 of the translated mRNA [24]

Summary

Transduction Kinases*

With molecular masses of 46 and 45 kDa were purified from EGF-stimulated A431 cells [8].Biochemical and immunolog-. The purified MAPKK was shown to phosphorylate the regulatory tyrosine and threonineresidue of the. From the Departments of $Pharmacology and §Biochemistry, University of Washington, Seattle, Washington 98195 native MAPK causing its full activation (€49).This restricted substrate specificity found for the MAPKK [5, 8] may be of particular significance in determining the specificity of the Mitogen-activatedprotein (MAP)kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that phosphorylate and activate MAP kinases signaling cascade. Slow autophosphorylation of MAPKK on serine, threonine, and tyrosine residues [8,9] confirmed that this enzyme belongs to the group of dual specificity protein kinases as suggested in several other reports [11,12,13,14,15]. We report the cloning of two forms of cDNA that encode this

EXPERIMENTAL PROCEDURES

RESULTS

YKK PYA