Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Argl487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Ai-gGly-Ile-Tyr-Arg1500, located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of CAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type I1 regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-( 11 89-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(l189-1525). Binding studies with PKA and Keywords: non-structural protein 3 ; protein phosphorylation ; CAMP-dependent protein kinase; chaperone.Hepatitis C virus (HCV) was identified as the major cause of non-A, non-B liver infections [ l ] that result in more than 50% of the investigated cases of chronic liver disease that lead to cirrhosis and hepatocellular carcinoma [2]. The mechanism by which HCV gene products exert their pathogenic effects is not understood. However, there are some indications that the interaction of viral antigens with intracellular proteins, after infection of the cell, could result in viral diseases of chronic nature. Since protein phosphorylation is an important step in the regulation of cell metabolism, differentiation and tumor promotion, the disturbance of this post-translational modification of proteins is often considered to be a possible mechanism for pathogenic pathways that are not understood. Examples in the literature include a number of viral antigens that interfere with intraCorrespondence to P.

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.

We describe in vitro tyrosine phosphorylation of the C‐terminal 334 amino acids of ras‐GTPase‐activating protein (ras‐GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N‐terminal domain of ras‐GAP Tyr‐460, which is considered to be the major phosphorylation site of ras‐GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF‐R compared to p60c‐src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF‐R) or increased (in case of p60c‐src) phosphorylation of the C‐terminal 334 amino acids of ras‐GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be ‐ independent from the presence of SH2 or SH3 domains ‐ triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr‐460.

Treatment of A431 cell membranes with trypsin or Streptomyces griseus proteinase results in degradation of the EGF-R and the concomitant generation of an active kinase with a molecular mass of 42 kDa (42 kDa kinase). To investigate the biochemical properties of the 42 kDa kinase, the EGF-R was immunoaffinity-purified from the A431 cell membranes and the kinase proteolytically generated. The proteolysis of EGF-R changes both the Vmax and the Michaelis constants of substrates. These substrates determine the extent of the changes of the parameters. The 42 kDa kinase is less responsive to polyions as regulators of kinase activity and is less efficiently inhibited by genistein and tyrphostin. The experiments described here point to a role of the extracatalytic domains in determining the substrate specificity and regulation of kinase activity.