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Autophosphorylation-dependent protein kinase(auto-kinase)
was found existing in pig brain and liver in 1987 by Yang et al.
with a unique property that its activity is regulated by
autophosphorylation.Auto-kinase is a cyclic nucleotide- and
calcium-independent protein kinase, and its molecular weight is
36kDa. Subsequent studies revealed that auto-kinase is a
multisubstrate protein kinase which can act on several key
regulatory proteins and enzymes with the following substrate
consensus sequence motif:R-X-(X)-S*/T*-X3-S/T (where * is the
kinase target site). However, the gene of auto-kinase has not
yet been cloned and therefore, the founding of auto-kinase as a
new protein kinase is still controversial. Furthermore, the
autophosphorylation site(s) of auto-kinase, which play(s) a
critical role in regulating its activity, has(have) not yet been
identified. To elucidate these points, we purified auto-kinase
was subjected to N-terminal and internal partial sequencing.
Three segments of partial amino acid sequence of auto-kinase
(VDGGAKTSDKQKKKAXMTDE, EKLRTIV and LQNPEI/KLTP/FI) were
successfully obtained. These sequences were perfectly matched
with those of a protein kinase named gamma-p21-Cdc42/Rac-
activated kinase(PAK2), which was identified from human placenta
cDNA library by Martin et al. in 1995, when mapped data bank.
This, together with our finding that auto-kinase can be
specifically recognized by an anti-peptide antibody raised
against the 14-amino acid C-terminal peptide of human PAK2
demonstrated that auto-kinase is a pig homologue of human PAK2.
A synthetic peptide EQSKRSTMVGTPYWMAPEVVTRK (STM peptide)
corresponding to amino acid residue 397-419 of human PAK2, which
possesses the substrate consensus sequence motif for auto-
kinase, can be efficiently phosphorylated by auto-kinase at
threonine residue. Moreover, the tryptic phosphopeptide map of
32P-STM peptide phosphorylated by auto-kinase is identical to
that of 32P-autophosphorylated tryptic phosphopeptides by manual
Edman degradation. These results demonstrated that Thr403
(EQSKRSTMVGTPYWMAPEVVTRK, * indicates the phosphorylation site,
according to the sequence of human PAK2) is the only
autophosphorylation site of auto-kinase.
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