Abstract:
Dynamic cellular activity is fundamental to all life. Virtually all life processes, are
modulated by the reversible phosphorylation of proteins, mediated by protein kinases and
phosphoprotein phosphatases, respectively. This thesis focuses on three enzymes,
namely: phosphoprotein phosphatase 1, phosphoprotein phosphatase 2A and protein
tyrosine phosphatase-1B. Temporal variations in the expression of the enzyme proteins
were examined in the human acute promyelocytic leukaemic cell line, HL-60. The cells
were induced to differentiate along the macrophage pathway using phorbol-12-myristate-
13-acetate and along the granulocytic pathway using dimethyl sulfoxide, all-trans retinoic
acid and 9-cis retinoic acid. Modulation of the rhythmic patterns of protein and
messenger RNA was monitored in the absence and presence of inducing agents.
Expression of protein in cell extracts prepared at various time intervals was determined
by western immunoblotting, while mRNA expression was assessed by northern blotting
and RT-PCR. The probe used for northern blotting was generated during the RT-PCR
procedure. In addition, PTP-1B mRNA was cloned into an expression vector to produce
recombinant protein.
Results indicate that the expression of phosphoprotein phosphatase 1, phosphoprotein
phosphatase 2A and protein tyrosine phosphatase-1B protein is dynamically regulated in
proliferating HL-60 cells and modulated after being induced to differentiate along either
the macrophage or granulocytic pathway. Similar changes were also noted with PTP-1B
mRNA when using northern blot analysis. Using molecular cloning techniques, PTP-1B
mRNA was successfully cloned into pGex-4T-1 expression vector to produce
recombinant PTP-1B protein, which was checked by sequence and western blot analysis.
