Ecto-protein kinase activities in normal and transformed cells

Area of Science:

• Cellular Biology
• Virology
• Biochemistry

Background:

• Rous sarcoma virus (RSV) transformation alters cellular protein phosphorylation.
• Protein kinases (PK) play crucial roles in cell signaling and regulation.
• Investigating cell surface protein phosphorylation provides insights into viral transformation.

Purpose of the Study:

• To identify and characterize protein kinases present on the surface of RSV-transformed cells.
• To investigate the phosphorylation sites and substrates of these surface kinases.
• To compare the properties of surface kinases with known viral and cellular kinases.

Main Methods:

• Incubation of intact chicken cells (uninfected and RSV-transformed) with [gamma-32P]ATP.
• Radioactive phosphorylation assays to detect kinase activity.
• Immunochemical analysis using antisera from RSV-tumor bearing rabbits (TBR-serum).
• PK assays and substrate phosphorylation experiments.
• Inhibition studies using diadenosine 5',5''-P1P4 tetraphosphate (Ap4A).

Main Results:

• Surface-located protein kinases were detected on both uninfected and RSV-transformed cells.
• Serine and threonine residues were identified as major phosphorylation sites.
• pp60v-src, a tyrosine kinase, was found on the surface of RSV-transformed cells.
• A distinct surface kinase, not cross-reactive with pp60v-src, was identified and released upon phosvitin incubation.
• pp60v-src phosphorylation was sensitive to Ap4A, while the phosvitin kinase activity was less sensitive.

Conclusions:

• RSV-transformed cells exhibit distinct surface protein kinase activities, including pp60v-src and a phosvitin kinase.
• These surface kinases phosphorylate serine and threonine residues.
• Differential sensitivity to Ap4A suggests unique functional roles for these surface kinases.
• Further research is needed to elucidate the functional significance and substrates of these cell surface PKs.