Humphries, William Arthur (2003) A study of a MARCKS-like protein Kinase C substrate in 'Lymnaea stagnalis' haemocytes. (MSc(R) thesis), Kingston University, .

Abstract

This study aimed to increase knowledge of Protein Kinase C (PKC) signalling and its major substrate Myristolated Alanine-Rich C Kinase Substrate (MARCKS) in the mollusc Lymnaea stagnalis haemocytes. Antibodies raised against peptides corresponding to the conserved regions ofPKC and MARCKS detected haemocyte proteins of similar molecular weight to those for PKC and MARCKS in a number of vertebrate systems. Upon challenge with phorbol-12-myristyrate-13-acetate (PMA), an increase in phosphorylation of both haemocyte PKC and MARCKS-like protein was observed, with maximal phosphorylation occurring at l Omin for PKC and l Smin for MARCKS-like protein was seen. Bacterial lipopolysaccharide (LPS) was also shown to stimulate haemocyte PKC and MARCKS-like protein, maximal phosphorylation occurred at 5min. The PKC inhibitors GF109203X and Calphostin C blocked these PMA and LPS mediated responses. PKC and MARCKS-like protein were also identified for the first time in a molluscan system using immunocytochemistry. Phagocytosis assays using the PKC inhibitors GFI09203X and Calphostin C showed that Calphostin C is a more potent inhibitor of phagocytosis (95.7% inhibited) than GF109203X (77.2% inhibited). These data show that a 'MARCKS-like' protein exists in L.stagnalis haemocytes, and that PKC may have a role in modulating its phosphorylation state, that PKC is involved in phagocytosis by haemocytes.

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