Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation.
Rotavirus NSP5 is a nonstructural protein that localizes in viroplasms of virus-infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced PAGE mobility. Here we show that NSP5 operates as an autoregulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. We developed an in vivo hyperphosphorylation assay in which two NSP5 mutant constructs are cotransfected. One of them, fused to an 11-aa tag, served as substrate whereas the other was used to map NSP5 domains required for activation. The activation and substrate activity could be uncoupled, demonstrating a hyperphosphorylation process in trans between the activator and substratum. This process involved dimerization of the two components through the 18-aa C-terminal tail. Phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. We present evidence of casein kinase 1alpha being the protein kinase responsible for this key step as well as for the consecutive ones leading to NSP5 hyperphosphorylation.
Eichwald
Catherine
C
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
Jacob
Germaine
G
Muszynski
Bartosz
B
Allende
Jorge E
JE
Burrone
Oscar R
OR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047389:Casein Kinase I / Q000378:metabolism*; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D019281:Dimerization; D000882:Haplorhini; D066298:In Vitro Techniques; D008954:Models, Biological; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012694:Serine; D047389:Casein Kinase I