Abstract
The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.
Highlights
Protein kinase (PK)1 D1 is a member of an evolutionarily conserved serine/threonine kinase family that in mammals has two other members, PKD2 and PKD3 [1,2,3]
The protein kinase C (PKC) isoform that mediates PKC activation can differ between different cell types; PKC␦ controls protein kinase D1 (PKD1) activation by oxidative stress in HeLa cells [16], whereas PKC⑀ is necessary for G protein-coupled receptor stimulation of PKD1 in Swiss 3T3 cells [17]
PKD1 catalytic activity in vivo can be monitored using antisera generated against a phosphoserine 916 peptide that selectively recognizes PKD1 molecules autophosphorylated on Ser-916
Summary
Protein kinase (PK) D1 is a member of an evolutionarily conserved serine/threonine kinase family that in mammals has two other members, PKD2 and PKD3 [1,2,3]. In this minimal model the phosphorylation of Ser-744/Ser-748 is sufficient for PKD1 stimulation [13, 20, 21] One basis for this conclusion is that a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active in Cos and HeLa cells. The PKC isoform that mediates PKC activation can differ between different cell types; PKC␦ controls PKD1 activation by oxidative stress in HeLa cells [16], whereas PKC⑀ is necessary for G protein-coupled receptor stimulation of PKD1 in Swiss 3T3 cells [17]. There is evidence for divergence in PKD1 regulation by different stimuli, as in HeLa cells the phosphorylation of tyrosine 463 was important for PKD1 activation in response to oxidative stress but not for PKD1 activation by a receptor-tyrosine kinase, the platelet derived growth factor receptor [27]
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