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The SD is characterized by fifteen YxxP motifs, ten of which can be efficiently phosphorylated by Src [15].
We found that His-OsPUB15C-1 could be efficiently phosphorylated by GST-PID2K, whereas His-OsPUB15N was not.
Here, we report the discovery that ubiquitinPhospho-Ser65 can prime Parkin to be efficiently phosphorylated by PINK1 at Ser of its Ubl domain, which in turn leads to maximal activation of Parkin E3 ligase activity.
Briefly, 50 μl of cell lysate was added to and incubated in wells pre-coated with a substrate peptide corresponding to the amino acid sequence surrounding mouse insulin receptor substrate 1 (IRS-1) Ser, which contains the serine residue that can be efficiently phosphorylated by AMPK.
The sequence around Ser does not conform to the canonical motif for CDPK substrates, because it lacks both the basic residue at P−3 and the hydrophobic residue at P−5, but nonetheless was shown to be efficiently phosphorylated by crude tomato extracts [ 33] and also recombinant CDPKs [ 27].
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Studies by Haystead and colleagues have indicated that peptides modelled on ROCK-I phosphorylation sites of MYPT1 and ZIPK are efficiently phosphorylated by the kinase (Hagerty et al, 2007).
T20 was efficiently phosphorylated by recombinant S6K in vitro, and systemic S6K activation (measured by phosphorylation at T398, corresponding to T389 in human S6K) was reduced in flies that had received infections with Toll or imd agonists.
GST-DCX was efficiently phosphorylated by both Rho-kinase-cat and PKN-cat.
We confirmed that CRMP-2 and DCX were efficiently phosphorylated by PKN in vitro.
Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro.
When we incubated recombinant hCdc14A with Plk1, Cdc14A was efficiently phosphorylated by Plk1 in vitro (Fig. 5F).
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