Identification of -R-X-(X)-S/T-X₃-S/T- as consensus sequence motif for autophosphorylation-dependent protein kinase

To identify consensus sequence motif for a new family of protein kinase termed autophosphorylation-dependent protein serine/threonine kinase (auto- kinase), we have tested several synthetic peptides. The well established protein serine/threonine kinases such as cAMP-dependent protein kinase, Ca²⁺/calmodulin-dependent protein kinase (CaM-kinase), and protein kinase C were found to be inactive toward phosphorylation of syntide-3 (RPRPASVPPSPSLSRHA), which turned out to be an excellent substrate only for auto-kinase, indicating that syntide-3 is a specific substrate for auto- kinase. Modification of syntide-3 to become RPRPASVPPS/T did not affect the activity of auto-kinase. By contrast, auto-kinase became rather or almost inactive when the peptide was modified to become RPRPASVPPA/G/F/K/R/D/E/Y, indicating that amino acid number 10 in syntide-3 is crucial to the sequence motif recognized by auto-kinase. Phosphorylation of myelin basic protein (MBP) by auto-kinase revealed that auto-kinase predominantly phosphorylates MBP on one particular site with RT-T(p)HYGS as the phosphorylation site sequence, which could not be phosphorylated by any other reported MBP kinases including cAMP-dependent protein kinase, CaM-kinase, protein kinase C, mitogen-activated protein kinase, and kinase FA/GSK-3. Taken together, the results provide initial evidence that -Arg-X-(X)-Ser/Thr-X₃-Ser/Thr- may represent a unique consensus sequence motif specifically recognized by autophosphorylation-dependent protein kinase, a new family of multisubstrate/multifunctional protein serine/threonine kinase.