Autophosphorylation properties of inactive and active JNK2.
The c-Jun N-terminal kinases (JNKs) are ubiquitous proteins that phosphorylate their substrates, such as transcription factors, in response to physical stress, cytokines or UV radiation. This leads to changes in gene expression, ensuing either cell cycle progression or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signalling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a recent chemical genetics investigation has confirmed that JNK transient activation leads to cellular proliferation, whereas a sustained one is pro-apoptotic. Here we investigate the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in glioma cells. Our results indicate that purified JNK2 from transfected nonstressed 293T cells is a mixture of the mono-sites pThr183 and pTyr185 of its activation loop and of pThr386 along its unique C-terminal region. Upon UV stimulation, its phosphorylation stoichiometry is upregulated on the activation loop, generating a mixture of mono-pTyr185 and the expected dual-pThr183/pTyr185 species, with the pThr386 specie present but unaltered respect to the basal conditions.
Pimienta
Genaro
G
Inflammation and Infectious Diseases Center, Burnham Institute for Medical Research, La Jolla, California 92037 USA.
Ficarro
Scott B
SB
Gutierrez
Gustavo J
GJ
Bhoumik
Anindita
A
Peters
Eric C
EC
Ronai
Ze'ev
Z
Pascual
Jaime
J
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D013058:Mass Spectrometry; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D007527:Isoenzymes; D010748:Phosphopeptides; D013912:Threonine; D014443:Tyrosine; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8