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Serine‐23 Is a Major Protein Kinase A Phosphorylation Site on the Amino‐Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins
Author(s) -
Sihag Ram K.,
Jaffe Howard,
Nixon Ralph A.,
Rong Xianhui
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1999.0720491.x
Subject(s) - map2k7 , casein kinase 2 , biochemistry , protein kinase a , phosphorylation , casein kinase 2, alpha 1 , cyclin dependent kinase 2 , protein subunit , mitogen activated protein kinase kinase , protein phosphorylation , c raf , kinase , biology , microbiology and biotechnology , threonine , chemistry , serine , gene
: We have shown previously that phosphate groups on theamino‐terminal head domain region of the middle molecular mass subunit ofneurofilament proteins (NF‐M) are added by second messenger‐dependent proteinkinases. Here, we have identified Ser 23 as a specific proteinkinase A phosphorylation site on the native NF‐M subunit and on two syntheticpeptides, S 1 ( 14 RRVPTETRSSF 24 ) andS 2 ( 21 RSSFSRVSGSPSSGFRSQSWS 41 ), localizedwithin the amino‐terminal head domain region. Ser 23 was identifiedas a phosphorylation site on the 32 P‐labeled α‐chymotrypticpeptide that carried >80% of the 32 P‐phosphates incorporatedinto the NF‐M subunit by protein kinase A. The synthetic peptidesS 1 and S 2 were phosphorylated 18 and two times moreefficiently by protein kinase A than protein kinase C, respectively. Neitherof the peptides was phosphorylated by casein kinase II. The sequence analysesof the chemically modified phosphorylated serine residues showed thatSer 23 was the major site of phosphorylation for protein kinase A onboth S 1 and S 2 peptides. Low levels of incorporation of 32 P‐phosphates into Ser 22 , Ser 28 , andSer 32 by protein kinase A were also observed. Protein kinase Cincorporated 32 P‐phosphates into Ser 22 ,Ser 23 , Ser 25 , Ser 28 , Ser 32 , and athreonine residue, but none of these sites could be assigned as a major siteof phosphorylation. Analyses of the phosphorylated synthetic peptides byliquid chromatography‐tandem mass spectrometry also showed that protein kinaseA phosphorylated only one site on peptide S 1 and that ions with upto four phosphates were detected on peptide S 2 . Analysis of thedata from the tandem ion trap mass spectrometry by using the computer programPEPSEARCH did not unequivocally identify the specific sites of phosphorylationon these serine‐rich peptides. Our data suggest that Ser 23 is amajor protein kinase A‐specific phosphorylation site on the amino‐terminalhead region of the NF‐M subunit. Phosphorylation of Ser 23 on the NF‐M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.