The Role of Membrane Protein Phosphorylation in Mitogen and Interleukin Four Mediated Activation of Murine B Lymphocytes

Beattie, Valerie Ann (1987) The Role of Membrane Protein Phosphorylation in Mitogen and Interleukin Four Mediated Activation of Murine B Lymphocytes. MSc(R) thesis, University of Glasgow.

Abstract

Activation of lymphocytes by the mitogens phytohaemagglutinin (PHA), 12-o-tetradecanoylphorbol-13-acetate (TPA) and anti-Ig has been observed to result in phosphorylation of a number of celluilar proteins. Lymphocyte activation by anti-Ig or TPA results in an increase in tyrosine phosphorylation of human B cell membrane proteins, thus implicating tyrosine kinase activities in the transmission of lymphocyte activation signals. In addition to the increase in phosphotyrosine levels upon activation by TPA, there is a concomitant increase in phosphoserine content due to the activation of protein kinase C. Lipopolysaccharide (LPS) causes translocation of protein kinase C from the cytoplasm to the plasma membrane and as lipid A, the free lipid portion of LPS, activates protein kinase C the possibilty arises that LPS, like TPA, activates protein kinase C directly. Plasma membrane was prepared from resting and LPS activated murine splenic B cells and enzyme marker assays indicated that the isolation procedure adopted was successfully enriching for plasma membrane. A comparision of total membrane protein phosphorylation in resting and LPS activated murine splenic B cells was carried out to determine if activation of these cells by LPS involved protein phosphorylation. Of the five samples examined, only 2 exhibited an increase in total membrane protein phosphorylation after a 24 hour incubation, in the presence of LPS, relative to resting B cell membranes. However, after a 48 hour incubation all five samples exhibited elevated levels of total membrane protein phosphorylation. Thus, activation of murine splenic B cells by LPS does appear to involve protein kinase activities. Phosphoamino acid analysis of resting and LPS activated B cell plasma membrane proteins was carried out in an attempt to identify the phosphoamino acid(s) involved in this increase in total membrane protein phosphorylation. The results of this phosphoamino acid analysis were variable and so it was not possible to determine clearly which phosphoamino acids were responsible for the increase observed. A 47Kd protein was identified by SDS-PAGE analysis of resting and LPS activated membrane protein profiles and this protein appeared to be more heavily phosphorylated upon LPS activation. Although both phosphoserine and phosphotyrosine were identified in this protein, it was not possible to accurately quantitate the individual phosphoamino acids and therefore determine which phosphoamino acid was responsible for the apparent increase in phosphorylation. Antigen specific activation of lymphocytes involves the action of T cell derived growth and differentiation factors, lymphokines. As the interleukin-2 receptor has been demonstrated to be phosphorylated by protein kinase C the possible involvement of protein phosphorylation in the action of interleukin-4 was investigated. As LPS activated B cells have been reported to express elevated numbers of interleukin-4 receptors the effect of interleukin-4 on membrane protein phosphorylation was investigated for both resting and LPS activated B cell membranes. No consistent alteration in the total membrane protein phosphorylation was observed for either resting or LPS activated B cell membrane, in response to IL-4. This observation did not eliminate the possibility that there were alterations in phosphorylation occurring at the level of individual proteins. In view of this membrane protein phosphorylated in the presence of IL-4 was examined by SDS-PAGE and compared to the results obtained from membrane protein phosphorylated in the absence of IL-4. While no alteration in phosphoprotein profile was observed, the possibilty remains that the level of individual phosphoamino acids within a given protein is changing although the total protein phosphorylation levels apparently remain unchanged. The small number of interleukin-4 receptors estimated to occur on a B cell may explain the lack of detectable response, at the biochemical level, in this system and more sensitive detection methods may be required to identify any changes occurring.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: Biochemistry
• Date of Award: 1987
• Depositing User: Enlighten Team
• Unique ID: glathesis:1987-77593
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77593