Plant pattern recognition receptors (PRRs) perceive microbial and endogenous molecular patterns to activate immune signaling. The cytoplasmic kinase BIK1 acts downstream of multiple PRRs as a rate-limiting component, whose phosphorylation and accumulation are central to immune signal propagation. Previous work identified the calcium-dependent protein kinase CPK28 and heterotrimeric G proteins as negative and positive regulators of BIK1 accumulation, respectively. However, mechanisms underlying this regulation remain unknown. The plant U-box proteins PUB25 and PUB26 are homologous E3 ligases that mark BIK1 for degradation to negatively regulate immunity. The heterotrimeric G proteins inhibit PUB25/26 activity to stabilize BIK1, whereas CPK28 specifically phosphorylates conserved residues in PUB25/26 to enhance their activity and promote BIK1 degradation. Interestingly, PUB25/26 specifically target non-activated BIK1, suggesting that activated BIK1 is maintained for immune signaling.

Q9LT79; At3g19380; Q9FXA4; At1g49780;

U-box domain-containing protein 25; Plant U-box protein 25; RING-type E3 ubiquitin transferase PUB25; PUB25; U-box domain-containing protein 26; Plant U-box protein 26; RING-type E3 ubiquitin transferase PUB26; PUB26;

Phospho-PUB25/26(T95/T94) Antibody detects endogenous levels of PUB25 when phosphorylated at Threonine 95 or PUB26 when phosphorylated at Threonine 94.

Arabidopsis thaliana(pT95); Zea mays(pT98, Uniport: A0A1D6HA67); Glycine max (pT69, Uniport: C6T8L8); 

Rabbit

WB 1:1000-1:2000

46kDa(Calculated).;

Store at -20 °C. Stable for 12 months from date of receipt.

WB 1:500-1:2000 

Figure 5. CPK28 Phosphorylates PUB25/26 to Enhance BIK1 Turnover and Dampen Immunity.

(B) CPK28, but not BIK1, phosphorylates PUB25^T95. Kinase assays were performed in vitro by incubating GST-PUB25 or GST-PUB25^T95A with HIS-BIK1, HIS-BIK1^K105E, and GST-CPK28. Protein phosphorylation was detected by anti-pT94/95 immunoblots. The fast-migrating band across all lanes was caused by background signal. Purified recombinant proteins were stained with Coomassie Brilliant Blue (CBB).

(C) Flg22 induces PUB25^T95 phosphorylation in a manner partially dependent on CPK28. Protoplasts of the indicated genotypes were transfected with the indicated constructs and treated with or without 1 μM flg22 for 10 min before total protein was extracted. PUB25-FLAG protein was enriched by affinity purification, and PUB25^T95 phosphorylation was detected by immunoblot using anti-pT94/95 antibodies (upper panel). Total PUB25-FLAG and CPK28-HA in the input were detected by anti-FLAG and anti-HA immunoblot, respectively (lower panels). Values indicate relative amounts of phosphorylated protein normalized to total PUB25-FLAG. Ponceau S staining of Rubisco indicates equal loading. Numbers indicate arbitrary units of PUB25^T95 phosphorylation calculated from densitometry measurements normalized to total PUB25-FLAG protein (sum of upper and lower bands).

(D) Flg22 enhances CPK28 kinase activity in plants. N. benthamiana plants transiently expressing CPK28-FLAG were treated with flg22 or H₂O 20 min prior to protein isolation. CPK28-FLAG was then affinity-purified and incubated with the recombinant GST-PUB26 protein in the in vitro kinase buffer. PUB26 phosphorylation was detected by anti-pT94/95 immunoblots. Purified GST-PUB26 was stained with Coomassie Brilliant Blue (CBB).

Jinlong Wang. ect. A Regulatory Module Controlling Homeostasis of a Plant Immune Kinase. Molecular Cell. Volume 69, Issue 3 p493-504.e6February 01, 2018.

DOI:https://doi.org/10.1016/j.molcel.2017.12.026