Abstract
A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.
Highlights
The protein kinase C (PKC)1 family is made up of at least 11 phospholipid-dependent serine/threonine kinases [1,2,3,4]
Identification of a Protein That Interacts with PKC—A twohybrid screen was initiated using the catalytic domain of protein kinase C (PKC)I as a bait
Clones were considered to have scored positively if they grew on plates lacking histidine and containing 3-AT, if they turned blue on 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal) plates, and if they grew on plates lacking uracil but failed to grow in 5-fluoroorotic acid (5-FOA), which is toxic if URA3 is expressed. 4 ϫ 106 independent transformants from a mouse embryo library were screened using this stringent approach
Summary
Plasmids—HA-tagged PKCI, PKC␣, and PKC␦ plasmids were generous gifts from Dr Shun’ichi Kuroda [10], Dr Peter Parker [16], and Dr Weiqun Li [17], respectively. The kinase domains of PKCI (residues 291– 673), PKCII (residues 291– 673), and PKC␦ (residues 343– 674) were used as baits in a yeast two-hybrid approach and were individually cloned into the SalI/SpeI sites of pPC97AD (Life Technologies, Inc.; kindly provided by Dr Marc Vidal, Dana-Farber Cancer Institute). A kinase dead PKK K51R mutant, was created using polymerase chain reaction-based mutagenesis and confirmed by sequencing. The expression of bait plasmids was confirmed by Western.
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