The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1.
The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1(T463A)-CFP and AtSERK1(3T-->A)-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.
Shah
Khalid
K
Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands.
Russinova
Eugenia
E
Gadella
Theodorus W J
TW
Willemse
Joost
J
De Vries
Sacco C
SC
D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D017931:DNA Primers; D004705:Endocytosis / Q000502:physiology*; D004735:Energy Transfer; D005453:Fluorescence; D016366:Open Reading Frames; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism*
D029681:Arabidopsis Proteins; D017931:DNA Primers; D011494:Protein Kinases; C440035:SERK1 protein, Arabidopsis; C090240:KAPP protein, Arabidopsis; D010749:Phosphoprotein Phosphatases