Figure 2.

Detection of polyribosome-bound kinase activity. In vitro phosphorylation assay of PRBP (10 μg per lane) prepared from cerebral cortical synaptoneurosomes from 12-day-old rats (24). This fraction includes ribosome-bound kinases as well as substrates. Incorporated ³³P was detected by PhosphorImaging. The activity of the polyribosome-bound kinases was not blocked by addition of specific inhibitor peptides against PKC (fragment 19–36, Sigma), PKA (PKI, Sigma), PKG (Arg-Lys-Arg-Ala-Arg-Lys-Glu, Sigma), or CaMKII (fragment 290–309, Sigma), each 2 μg per assay, or inhibitors against protein kinase R (100 mM 2-aminopurine = 2-AP), casein kinase II (increasing concentrations of heparin), PKC (10 μM calphostin C). GF109203X (10 μM), an inhibitor of PKC and p90rsk, had a small inhibitory effect. The kinase activities could be blocked completely by capturing divalent cations (Mg²⁺, Ca²⁺) with 50 mM EDTA or heating the fraction to 70°C for 5 min and inhibited almost completely by 10 μM staurosporine, a nonspecific inhibitor of Ser/Thr kinases.