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. 2018 Jun 5;30(7):1543–1561. doi: 10.1105/tpc.17.00981

Figure 8.

Figure 8.

Both RLCK VII-4 and MPK4 Phosphorylate MEKK1 at Ser-603.

(A) Pattern-triggered phosphorylation of MEKK1 at the C terminus. Protoplasts expressing C-MEKK1K361M-HA were treated with flg22, elf18, or chitin. Total protein was fractionated by SDS-PAGE containing Phos-tag acrylamide and detected by immunoblotting with anti-HA antibody.

(B) Ser-603 is the major phospho-site in MEKK1 C terminus MEKK1 upon chitin and flg22 induction. C-MEKK1K361M-HA (WT) or C-MEKK1K361M/S603A-HA (S603A) was expressed in protoplasts and total protein was detected by immunoblotting with anti-HA antibody.

(C) Chitin-triggered phosphorylation of MEKK1 at Ser-603. Stable transgenic seedlings expressing MEKK1-FLAG were sprayed with chitin. MEKK1-FLAG was affinity purified with anti-FLAG antibody and detected by immunoblotting with anti-pSer603 antibodies for MEKK1 phosphorylation.

(D) Chitin-triggered phosphorylation of Ser-603 is impaired in the rlck vii-4 sextuple mutant. Protoplasts of the indicated genotypes transfected with the C-MEKK1 K361M-HA constructs containing a wild-type Ser603 (WT) or a Ser603Ala substitution (S603A) were treated with chitin. The C-MEKK1K361M-HA protein was immunoprecipitated with anti-HA antibodies, and Ser-603 phosphorylation was detected by immunoblotting with anti-pSer603 antibodies. Total C-MEKK1K361M-HA protein was detected by immunoblotting with anti-HA antibody. Numbers indicate arbitrary densitometry units of pSer603 normalized to total C-MEKK1 K361M-HA protein.

(E) CERK1-activated PBL19 phosphorylates the MEKK1 C-terminal tail in vitro. GST-MEKK1-T was incubated with PBL19-His and/or CERK1-KD-His (CERK1-C-His) proteins, and MEKK1 phosphorylation was detected by immunoblotting with anti-pSer603 antibodies.

(F) Flg22- or chitin-triggered Ser-603 phosphorylation of MEKK1 is impaired in the summ2-8 mpk4 mutant. Numbers indicate arbitrary densitometry units of pSer603 normalized to total C-MEKK1 K361M-HA protein.

(G) Activated MPK4 phosphorylates MEKK1 in vitro. Protoplasts expressing MPK4-FLAG were treated with (+) or without (−) flg22. MPK4-FLAG proteins were affinity-purified with anti-FLAG antibodies and incubated with GST-MEKK1-T protein in kinase buffer, and MEKK1 phosphorylation was detected by immunoblotting with anti-pSer603 antibodies.