[논문]Serine 389 phosphorylation of 3-phosphoinositide-dependent kinase 1 by UNC-51-like kinase 1 affects its ability to regulate Akt and p70 S6kinase

Kim, Kidae; Park, Sung Goo; Park, Byoung Chul; Kim, Jeong-Hoon; Kim, Sunhong

doi:10.5483/bmbrep.2020.53.7.299

[국내논문] Serine 389 phosphorylation of 3-phosphoinositide-dependent kinase 1 by UNC-51-like kinase 1 affects its ability to regulate Akt and p70 S6kinase 원문보기

BMB reports, v.53 no.7, 2020년, pp.373 - 378

Kim, Kidae (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology) , Park, Sung Goo (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology) , Park, Byoung Chul (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology) , Kim, Jeong-Hoon (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology) , Kim, Sunhong (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)

Abstract ▼ AI-Helper

Phosphorylation of the signaling component by protein kinase often leads to a kinase cascade or feedback loop. 3-Phosphoinositide-dependent kinase 1 (PDK1) signaling pathway diverges into various kinases including Akt and p70 S6 kinase (p70S6k). However, the PDK1 feedback mechanism remains elusive. Here, we demonstrated that UNC-51-like kinase (ULK1), an autophagy initiator kinase downstream of mechanistic target of rapamycin (mTOR), directly phosphorylated PDK1 on serine 389 at the linker region. Furthermore, our data showed that this phosphorylation affected the kinase activity of PDK1 toward downstream substrates. These results suggest a possible negative feedback loop between PDK1 and ULK1.

Keyword

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문제 정의

To our knowledge, this work is the first report demonstrating the phosphorylation of Ser389 in PDK1. Based on previous reports, a few hypotheses can be proposed: First, since Ser389 is located in the linker region between kinase and PH domains of PDK1, phosphorylation at this residue might induce conformational changes in PDK1 protein, leading to altered substrate recognition of the kinase, which is supported by the results suggesting that PDK1 kinase activity was not altered in S389A mutant (Fig.

가설 설정

3C). Next, we tested whether the activation of ULK1 affected the phosphorylation of PDK1. We used oligomycin A, which is known to induce energy deprivation, and rapamycin, which inhibits mTOR and induces subsequent increase in ULK1 activity.

제안 방법

3A). In order to investigate the characteristics of PDK1 phosphorylation at Ser389, we generated phospho-specific antibody that recognizes phosphorylated Ser389 of PDK1 (pS389 PDK1 antibody). HEK293T cells transfected with PDK1 alone or together with ULK1 were lysed and PDK1 proteins were immunoprecipitated using anti-Myc antibody.
The anti-HA immunoblot (bottom panel) was also conducted using the same whole cell lysates. (B) HEK293T cells transfected with HA-ULK1 WT or KI were lysed and ULK1 assays were performed using GST-PDK1 linker protein as a substrate as described in Materials and Methods. The phosphorylated proteins were visualized by autoradiography (top panel).
4A). First, the in vitro kinase assay was carried out to measure the activity of PDK1. It was intriguing that the activity of PDK1 S389A is very similar to wild type in vitro (Fig.
1D). To identify the domains of PDK1 interacting with ULK1, we generated the truncation mutants of PDK1 (Fig. 1E) and performed the GST pull-down assay. HA-ULK1 was bound to GST-PDK1 WT, KL and C but not PH (Fig.

대상 데이터

Anti-Flag M2 (F1804), anti-GST (G7781), anti-Tubulin (T6074), Oligomycin A (75351) and Rapamycin (R8781) were obtained from Merck (St. Louis, MO, USA). Anti-Myc (sc-40), anti-HA (sc-805) and anti-GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Louis, MO, USA). Anti-Myc (sc-40), anti-HA (sc-805) and anti-GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Myc (2278S), anti-Phospho-ULK1 Ser317 (6887S), anti-ULK1 (8054S), antiPhospho-Akt Ser473 (4060S), anti-Phospho-Akt Thr308 (4056S), anti-Akt (4691S), anti-Phospho-p70S6k Thr389 (9234S), antip70S6k (2708S) and anti-PDK1 (3062S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
Anti-Myc (sc-40), anti-HA (sc-805) and anti-GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Myc (2278S), anti-Phospho-ULK1 Ser317 (6887S), anti-ULK1 (8054S), antiPhospho-Akt Ser473 (4060S), anti-Phospho-Akt Thr308 (4056S), anti-Akt (4691S), anti-Phospho-p70S6k Thr389 (9234S), antip70S6k (2708S) and anti-PDK1 (3062S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Phospho-p70Sk Thr229 (MAB8964) was purchased from R&D systems (Minneapolis, MN, USA).
ULK1 inhibitor, SBI-0206965 (S7885) were obtained from Selleckchem (Houston, TX, USA). Anti-HA Magnetic Beads (88837), Glutathione Magnetic Agarose Beads (78601), Anti-c-Myc Magnetic Beads (88843), Anti-DYKDDDDK Magnetic Agarose (A36798) and Dynabeads^TM Protein G for Immunoprecipitation (10004D) were purchased from ThermoFisher and used for immunoprecipitation.

데이터처리

Statistical analysis was performed using Student’s t-test, and P-value ＜ 0.05 was considered significant; however, the calculated P value was 0.69.

성능/효과

In conclusion, our study suggests that Ser389 phosphorylation of PDK1 regulates its signaling function and the existence of a novel negative feedback loop between PDK1 and ULK1/ autophagy pathway.

후속연구

However, specific conditions might determine the direction of feedback. Further studies are needed to elucidate the mechanism underlying this novel feedback loop.

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