PMID- 23863167
OWN - NLM
STAT- MEDLINE
DCOM- 20131206
LR  - 20211021
IS  - 1470-8752 (Electronic)
IS  - 0300-5127 (Print)
IS  - 0300-5127 (Linking)
VI  - 41
IP  - 4
DP  - 2013 Aug
TI  - Pseudokinases from a structural perspective.
PG  - 981-6
LID - 10.1042/BST20130120 [doi]
AB  - The catalytic (C) subunit of PKA was the first protein kinase structure to be 
      solved, and it continues to serve as the prototype for the protein kinase 
      superfamily. In contrast, by comparing many active and inactive kinases, we 
      developed a novel 'spine' concept where every active kinase is composed of two 
      hydrophobic spines anchored to a hydrophobic F-helix. The R-spine (regulatory 
      spine) is dynamically assembled, typically by activation loop phosphorylation, 
      whereas the C-spine (catalytic spine) is completed by the adenine ring of ATP. In 
      the present paper, we show how the spine concept can be applied to B-Raf, 
      specifically to engineer a kinase-dead pseudokinase. To achieve this, we mutated 
      one of the C-spine residues in the N-lobe (N-terminal lobe), Ala481, to 
      phenylalanine. This mutant cannot bind ATP and is thus kinase-dead, presumably 
      because the phenylalanine ring fills the adenine-binding pocket. The C-spine is 
      thus fused. However, the A481F mutant is still capable of binding wild-type B-Raf 
      and wild-type C-Raf, and dimerization with a wild-type Raf leads to downstream 
      activation of MEK [MAPK (mitogen-activated protein kinase)/ERK 
      (extracellular-signal-regulated kinase) kinase] and ERK. The mutant requires 
      dimerization, but is independent of Ras and does not require enzymatic activity. 
      By distinguishing between catalytic and scaffold functions of B-Raf, we define 
      kinases as being bifunctional and show that, at least in some cases, the scaffold 
      function is sufficient for downstream signalling. Since this alanine residue is 
      one of the most highly conserved residues in the kinome, we suggest that this may 
      be a general strategy for engineering kinase-dead pseudokinases and exploring 
      biological functions that are independent of catalysis.
FAU - Taylor, Susan S
AU  - Taylor SS
AD  - Department of Biomedical Sciences, University of California San Diego, 9500 
      Gilman Drive, La Jolla, CA 92093, USA. staylor@ucsd.edu
FAU - Shaw, Andrey
AU  - Shaw A
FAU - Hu, Jiancheng
AU  - Hu J
FAU - Meharena, Hiruy S
AU  - Meharena HS
FAU - Kornev, Alexandr
AU  - Kornev A
LA  - eng
GR  - R37 AI057966/AI/NIAID NIH HHS/United States
GR  - AI57966/AI/NIAID NIH HHS/United States
GR  - R01 GM019301/GM/NIGMS NIH HHS/United States
GR  - R37 GM019301/GM/NIGMS NIH HHS/United States
GR  - GM19301/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Review
PL  - England
TA  - Biochem Soc Trans
JT  - Biochemical Society transactions
JID - 7506897
RN  - EC 2.7.- (Protein Kinases)
RN  - EC 2.7.11.1 (raf Kinases)
SB  - IM
MH  - Biocatalysis
MH  - Models, Molecular
MH  - Protein Conformation
MH  - Protein Kinases/chemistry/*metabolism
MH  - raf Kinases/metabolism
PMC - PMC4399968
MID - NIHMS677057
EDAT- 2013/07/19 06:00
MHDA- 2013/12/16 06:00
PMCR- 2015/04/16
CRDT- 2013/07/19 06:00
PHST- 2013/07/19 06:00 [entrez]
PHST- 2013/07/19 06:00 [pubmed]
PHST- 2013/12/16 06:00 [medline]
PHST- 2015/04/16 00:00 [pmc-release]
AID - BST20130120 [pii]
AID - 10.1042/BST20130120 [doi]
PST - ppublish
SO  - Biochem Soc Trans. 2013 Aug;41(4):981-6. doi: 10.1042/BST20130120.