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Trans‐phosphorylation Activates The Yeast Protein Kinase Kin2, An Ortholog Of Human Microtubule Affinity Regulating Kinase
Author(s) -
Ghosh Chandrima,
Sathe Leena,
Dey Madhusudan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.662.9
Subject(s) - map2k7 , kinase , biology , biochemistry , phosphorylation , mitogen activated protein kinase kinase , microbiology and biotechnology , protein kinase a , ask1 , protein phosphorylation , cyclin dependent kinase 2 , protein kinase domain , map kinase kinase kinase , mutant , gene
Yeast protein kinases Kin1 and its isoform Kin2 are orthologs of the human microtubule affinity‐regulating kinase (MARK), which are known to regulate multiple cellular processes, including cellular exocytosis and polarity. Recently, we show that Kin kinases regulate cellular protein homeostasis or proteostasis by an unknown mechanism. To decipher the role of Kin kinases in proteostasis network, we focused our studies on the Kin2 kinase. We observed that the N‐terminal half of Kin2 protein (residues 94–510, referred to here as the Kin2 mini ) fully complemented the Kin2 protein null strain. These data suggest that Kin2 mini plays a primary role in cellular processes. The subsequent mass spectrometric analysis of the Kin2 mini protein identified three phosphorylated peptides with five phospho‐acceptor residues. Mutations of only two phospho‐acceptor residues Y275 and T281 by non‐phosphorylatable alanines in a single protein ( i.e. , Kin2 mini ‐Y275A, T281A) completely abolished the kinase activity both in vivo and in vitro . These results suggest that phosphorylation of the residues Y275 and T281 are important for its kinase function. We also observed that substitution of the residue T281 by a phosphomimetic glutamate ( i.e. , Kin2 mini ‐Y275A, T281E) restored the Kin2 kinase function. These data suggest that T281 is the key phosphorylation residue for the Kin2 kinase function. Furthermore, we found that the kinase‐dead Kin2 mini ‐D248A mutant (D of the conserved catalytic HRD motif) was phosphorylated on the residue T281 in vivo and the recombinant glutathione S‐transferase (GST)‐fused Kin2‐T281E mutant protein, but not the GST‐Kin2 wild type protein, was an active kinase in vitro . Collectively, our data demonstrate that Kin2 kinase is activated by transphosphorylation on the residue T281 by an upstream kinase. Currently we are studying how an active Kin2 kinase initiates a signaling cascade to fine‐tune the proteostasis network. Characterization of the Kin‐kinase signaling pathway in yeast, we believe, will illuminate the inner workings of the MARK signaling pathway in human cells. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .