Properties and mechanism of action of viral and cellular tyrosyl protein kinases

Abstract

We have demonstrated the usefulness of several angiotensin analogs as substrates for a number of tyrosyl protein kinases of viral or cellular origin. Results of initial rate studies with pp60 scr at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 μ m and 0.24 μ m respectively and V max was 1.0 nmol/min/mg. The end-product ADP and the ATP analog, AMP-PNP, were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [ΔPhe 4]-angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. The available evidence allowed us to conclude that while pp60 src contained essential histidine and lysine residues in its active site, the kinase reaction does not involve a phosphoryl enzyme intermediate. Phosphorylation of the angiotensin peptides in vitro also has allowed us to demonstrate the presence of at least two tyrosyl protein kinases in the cytoplasm of normal rat liver cells. These kinases appear to be novel in that they are present in normal cells and are not stimulated by growth factors. Also, results of preliminary experiments indicate that these kinases are not immunologically related to the transforming gene products of Rous and Fujinami sarcoma viruses (unpublished observations). The identification of these new kinases represents another application of the angiotensin peptides as substrates for tyrosyl kinases (13). The results obtained do not exclude the possibility that there exist in rat liver other tyrosyl kinases that do not phosphorylate these particular peptide substrates. No attempt has been made to characterize tyrosyl kinases associated with the plasma membrane fraction. Although they repreent only a small fraction of the total activity of liver cells, the plasma membrane kinases have a relatively high specific activity. These kinases may be identical with growth factor receptor kinases previously identified in liver cell membranes (5). The most abundant tyrosyl kinase in rat liver cytoplasm has a molecular weight of 75,000 daltons and was found in cytosol and microsomal salt-wash fraction. The observation that the purified 75 Kd enzyme phosphorylates a 75 Kd protein on tyrosine residues suggests that the enzyme may possess autophosphorylating activity. A high molecular weight (greater than 160 Kd) kinase also was found in both the cytosol and microsomal salt-wash fraction. Since this high molecular weight material was very heterogeneous, we did not attempt to analyze its reaction products in an in vitro phosphorylation reaction. There is as yet no evidence to indicate whether the high molecular weight enzyme is an aggregated form of TPK 75 or whether it is an entirely different enzyme. Furthermore, it is not clear how these two kinases relate to those that were not extractable from microsomal membranes. Resolution of these issues will have to await further purification and characterization of the different enzymes. To summarize, rat liver cytoplasm may contain as many as three tyrosyl protein kinases: TPK 75, a high molecular weight enzyme, and an enzyme that associates tightly with microsomes. The available data do not allow us to conclude whether or not the association of tyrosyl kinases with ribosomes and endoplasmic reticulum is biologically significant. However, such an association is rather novel and suggests that tyrosyl kinases may play a role in regulating the activity of the protein synthetic machinery. Considerable evidence has been presented for the significance of phosphorylation of serine and threonine residues of ribosomal proteins and initiation factors in protein synthesis and cellular metabolic activities. Although there has not been any report of tyrosine-specific phosphorylation in these components, the involvement of phosphatases has not been adequately dealt with. In view of the findings presented in this report, these issues merit reevaluation.