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A Modified Immunoblot Method to Identify Substrates of Protein Kinases
Choong-Min Kang,Wan Jin Jahng,Robert N. Husson,이상희 한국미생물학회 2011 The journal of microbiology Vol.49 No.3
While protein kinases are key components in multiple cellular processes, efficient identification of cognate in vivo substrates remains challenging. Here we describe a powerful method to screen potential substrates of protein kinases by partial transfer of proteins from a 2D-PAGE gel to a Western blot membrane. This approach allowed precise pinpointing of candidate substrate spots in the 2D gel, and identifying physiological substrates of protein kinases in Mycobacterium tuberculosis.
Song, Taeksun,Song, Seung-Eun,Raman, Sahadevan,Anaya, Mauricio,Husson, Robert N. American Society for Microbiology 2008 Journal of Bacteriology Vol.190 No.6
<B>ABSTRACT</B><P>Mycobacterial SigE and SigH both initiate transcription from the <I>sigB</I> promoter, suggesting that they recognize similar sequences. Through mutational and primer extension analyses, we determined that SigE and SigH recognize nearly identical promoters, with differences at the 3′ end of the −35 element distinguishing between SigE- and SigH-dependent promoters.</P>
Phosphorylation Regulates Mycobacterial Proteasome
Tripti Anandan,Jaeil Han,Heather Baun,Seeta Nyayapathy,Jacob T. Brown,Rebekah L. Dial,Juan A. Moltalvo,Min-Seon Kim,양승환,Donald R. Ronning,Robert N. Husson,서주원,강충민 한국미생물학회 2014 The journal of microbiology Vol.52 No.9
Mycobacterium tuberculosis possesses a proteasome systemthat is required for the microbe to resist elimination by thehost immune system. Despite the importance of the proteasomein the pathogenesis of tuberculosis, the molecular mechanismsby which proteasome activity is controlled remainlargely unknown. Here, we demonstrate that the α-subunit(PrcA) of the M. tuberculosis proteasome is phosphorylatedby the PknB kinase at three threonine residues (T84, T202,and T178) in a sequential manner. Furthermore, the proteasomewith phosphorylated PrcA enhances the degradation ofIno1, a known proteasomal substrate, suggesting that PknBregulates the proteolytic activity of the proteasome. Previousstudies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactivenitrogen intermediates (RNIs) but increases resistance tohydrogen peroxide (H2O2). Here we show that PknA phosphorylationof unprocessed proteasome β-subunit (pre-PrcB)and α-subunit reduces the assembly of the proteasome complexand thereby enhances the mycobacterial resistance toH2O2 and that H2O2 stress diminishes the formation of theproteasome complex in a PknA-dependent manner. Thesefindings indicate that phosphorylation of the M. tuberculosisproteasome not only modulates proteolytic activity of theproteasome, but also affects the proteasome complex formationcontributing to the survival of M. tuberculosis underoxidative stress conditions.