Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

Abstract

The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

Interaction of Bruton's Tyrosine Kinase and Protein Kinase Cθ in Platelets

“…This is a novel finding, but because of the nonselectivity of BIM among the PKC isoforms it is not possible at this stage to assess whether Btk is specifically regulated by associated PKC. Precedence for the activation of a tyrosine kinase by PKC-mediated phosphorylation has been set by the demonstration that PKC␦ phosphorylates and activates the kinase c-Abl in vitro (39), PKC activates Fyn (40), and PKC␣, ␤1, ␤2, and ␥ are upstream of Syk (41). In other cell types, PKC isoforms have been shown to phosphorylate Btk, leading to an inhibition of Btk activity (13), and therefore our data are in contrast with these reports.…”

Crystal-Induced Neutrophil Activation: XI. Implication and Novel Roles of Classical Protein Kinase C

“…The PKC-mediated serine phosphorylation of the catalytic subunit of Syk has been previously shown to increase its in vitro kinase activity (62), although this activity has not been confirmed in recent studies with human platelets (49). We show that the PKCmediated serine phosphorylation of Syk is necessary for its MSU crystal-induced tyrosine phosphorylation because both Gö6976 and silencing PKC␣ diminish it.…”

Requirement of Syk‐phospholipase C‐γ2 pathway for phorbol ester‐induced phospholipase D activation in DT40 cells

“…In contrast, Borowski et al . reported that the 40 kDa catalytic domain of Syk, a partially degradated product, purified from rat spleen was phosphorylated and activated by PKC in vitro (Borowski et al 1998). Therefore, it is possible that in intact cells, an unknown modification by some factor(s) enabled Syk to receive direct phosphorylation and activation by PKC.…”