Fat location defines sensation

Tyrosine Phosphorylation of the BRI1 Receptor Kinase Occurs via a Post-Translational Modification and is Activated by the Juxtamembrane Domain

“…As part of these studies we have further examined the mechanism of BRI1 autophosphorylation in order to understand two aspects in particular: (1) the activating role of the juxtamembrane domain; and (2) the paradox of tyrosine autophosphorylation. With respect to the juxtamembrane domain, we showed in a previous study (Oh et al, 2009) that the BRI1 kinase domain, in the absence of the juxtamembrane domain, could autophosphorylate on serine residues but not on threonine or tyrosine residues. This was interesting because many of the sites of threonine and tyrosine autophosphorylation reside within the kinase domain, but yet are not autophosphorylated if the entire juxtamembrane domain is removed.…”

“…Membranes were blocked in a 2% (v/v) fish gelatin solution in phosphate-buffered saline (PBS; 5 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.4) before being incubated with primary antibodies, which were diluted as specified in PBS containing 0.1% (v/v) Tween-20 (PBST). Blots were probed with custom polyclonal antibodies that recognize BRI1 phosphotyrosine-831 (pY831; 1:1000 dilution), phosphotyrosine-956 (pY956; 1:1000 dilution), or phosphotyrosine-1072 (pY1072; 1:1,000 dilution; Oh et al, 2009), or phosphoserine-858 (pS858; 1:2,000 dilution), phosphothreonine-872 (pT872; 1:3,000 dilution), or phosphoserine-891 (pS891; 1:2,000 dilution; Oh et al, 2012b). Alternatively, blots were stained with ProQ Diamond phosphoprotein stain (Invitrogen, Grand Island, NY, USA) or Coomassie Brilliant Blue (CBB) as specified.…”

“…The resuspending pellet was then incubated at 30°C for 1 h, centrifuged at 24,000 g for 20 min, and the resulting supernatant was transferred to a new tube. The protein concentration was determined with Bio-Rad protein assay using BSA as a standard and the 2-DE analysis was performed as described (Oh et al, 2009). Total protein (300 μg) was loaded onto Immobiline™ DryStrip pH 3–10 (13 cm) strips and focused on the IPGphor IEF system for a total of 20 h (68 kVh) at 20°C.…”

“…In the present study, we produced a nested series of juxtamembrane domain truncations to determine which sequences were essential for the activation. With respect to our second point of interest, the paradox of tyrosine autophosphorylation emerged when BRI1 was shown to contain phosphotyrosine (Oh et al, 2009) but autophosphorylation on tyrosine in vitro had not been observed to occur (Friedrichssen et al, 2000; Oh et al, 2000; Wang et al, 2005a). One plausible explanation to reconcile these conflicting observations would be that autophosphorylation on tyrosine is a co-translational, not post-translational, modification.…”

“…Animal receptor kinases are predominantly tyrosine kinases while plant receptor kinases are generally classified as serine/threonine kinases. However, recent work suggests that at least some plant receptor kinases are dual-specificity kinases that can autophosphorylate on serine, threonine, and tyrosine residues (Karlova et al, 2009; Oh et al, 2009, 2010). One of the most thoroughly studied plant receptor kinases is Brassinosteroid Insensitive 1 (BRI1), which functions with its co-receptor, BRI1-associated receptor kinase 1 (BAK1), in brassinosteroid (BR) signaling (Vert et al, 2005; Gendron and Wang, 2007; Clouse, 2011).…”