Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation

Abstract

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation.

Keywords: autophosphorylation; dual-specificity; kinase; protein kinase D.

Figure 1

Protein kinase D displays autophosphorylation on Tyr residues. (A) Time course of PKD1 phosphorylation on Tyr residues by PKD1 in vitro. FLAG‐PKD1 was purified from HEK293 cells as described in the Materials and methods section, incubated with Mg2+.ATP for the indicated timepoints and assayed for Tyr phosphorylation via immunoblotting. (B) in vitro kinase assay (IVKA) for PKD1 autophosphorylation in presence of the indicated inhibitors. Quantification of two individual experiments is shown. Graphs represent mean ± SEM. (C) Progress curves of Syntide‐2 phosphorylation by PKD1. FLAG‐PKD1 purified from HEK293 cells was incubated with one of three different Syntide‐2 derived peptides; where Syn‐2S is a peptide containing Ser as phospho‐acceptor, Syn‐2T contains a Thr and Syn‐2Y contains a Tyr as phospho‐acceptor. (D) Activity of Y749F and kinase‐dead K612A mutants towards Syntide‐2. FLAG‐tagged PKD1 WT and K612A or Y749F mutants were incubated with Syn‐2 peptide and activity was measured in a radiometric kinase assay. (E) Autophosphorylation of PKD1 Y749F on Tyr residues. FLAG‐PKD1 WT and a Y749F mutant were incubated with Mg2+.ATP for the indicated time points and assayed for Tyr phosphorylation via immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean SEM.

Figure 2

Structural determinants for Tyr autophosphorylation. (A) PKD autophosphorylates on Tyr in cis and Tyr kinase activity is inhibited in presence of PS/PDB. GST‐tagged PKD WT and FLAG‐tagged PKD KD were incubated separately or together in presence or absence of ATP and allowed to react for 30′ at 30 °C. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (B) Time course of in vitro autophosphorylation by FLAG‐PKD1 purified from unstimulated or cells stimulated with PDB (500 nm, 10′). PKD1 was incubated with Mg2+.ATP for the indicated time points and assayed for Tyr and Ser‐738/742 phosphorylation via immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean ± SEM.

Figure 3

A Protein kinase D C705R mutant results in altered enzymatic properties. (A) Multiple sequence alignment of several reported dual‐specificity kinases that have substitutions for the conserved Arg in the HRD motif. Alignment was performed in Vector NTi using the alignX module. (B) Ser and Tyr autophosphorylation activities of PKD WT and the C705R mutant in presence or absence of PS/PDB containing SUVs. FLAG‐PKD1 WT and a C705R mutant were purified from HEK293 cells as described in the Materials and methods section, incubated with Mg2+.ATP for the indicated time points and assayed for Tyr and Ser‐738/742 phosphorylation via immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (C) Activities toward Syn‐2 of PKD WT and C705R in the inactive state of PKD. (D) Activities toward Syn‐2 of PKD WT and C705R after 60 min of prior autophosphorylation in presence of PS/PDB containing SUVs. A blot representing the phosphorylation state of PKD1 preparations used for C and D is shown in the right panel of Fig. 3D.

Figure 4

Protein kinase D associates with a factor in cells that impedes its autophosphorylation at Tyr residues. (A) Tyr phosphorylation of PKD after stimulation of HEK293 cells with H₂O₂ (10 mm, 10 min) in the presence of the indicated kinase inhibitors (5 μm). FLAG‐PKD1 was precipitated from cells and the Tyr phosphorylation state was assessed by immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (B) Tyr phosphorylation of PKD after stimulation with pervanadate (75 μm, 30 min) in the presence of the indicated kinase inhibitors (5 μm). FLAG‐PKD1 was precipitated from HEK293 cells and the Tyr phosphorylation state was assessed by immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (C) Autophosphorylation on Tyr residues of differentially purified PKD preparations. For details see text. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (D) Tyr autophosphorylation of MBP‐mPKD1‐CAT purified from bacteria. MBP‐mPKD1‐CAT was incubated with Mg2+.ATP for 45 min in presence or absence of PKD inhibitor or Calf Intestine Alkaline Phosphatase (CIP) and assayed for Tyr phosphorylation via immunoblotting. Quantification of three individual experiments is shown. Graphs represent mean ± SEM. (E) Expression of MBP‐mPKD1‐CAT in BL21‐Rosetta cells in presence or absence of the PKD inhibitor CRT 0066101. BL21 cells harboring pMALC2X‐MBP‐mPKD1‐CAT were grown and at OD600 of 0.6 the culture was induced with 0.5 mm IPTG and divided to either incubate with or without 5 μm CRT 0066101. Four hours post induction cell pellets were collected and analyzed for Tyr phosphorylation by immunoblotting.

Figure 5

Mechanisms that regulate PKD Tyr autophosphorylation. Modalities in which PKD can acquire Tyr specificity. PKD purified from resting cells displays potent Tyr autophosphorylation. Ser‐738/742 autophosphorylation is still at low levels in an unstimulated enzyme. Activation with PDB results in increased Ser‐738/742 autophosphorylation and decreased Tyr autophosphorylation activity. Mutation of Cys to Arg in the catalytic loop HCD motif results in loss of Tyr and Ser‐738/742 autophosphorylation in unstimulated conditions and an increase in PDB stimulated Ser‐738/742 autophosphorylation. Cellular autophosphorylation activity is likely suppressed by an inhibitory factor (X) associating with PKD.