c-abl is involved in the association of p53 and trk A.

The p53 tumour suppressor phosphoprotein associates with proteins involved in DNA replication, transcription, cell cycle machinery and regulation of its own expression. Recently it has been shown that p53 can also bind to trk A tyrosine kinase which is the receptor for nerve growth factor (NGF). This study demonstrates that p53 appears to associate with trk A via c-abl. Endogenous c-abl was detected when the trk A and p53 complex was immunoprecipitated from lysates of NGF stimulated NIH3T3 cells expressing trk A or NIH3T3 cells expressing trk A and a temperature sensitive p53 (val 135). Endogenous c-abl and trk A association was observed in NGF stimulated p53 negative fibroblasts transfected with trk A alone; suggesting that c-abl can independently bind to trk A in the absence of p53. Interestingly, association between endogenous p53 and trk A was not detected in NGF stimulated abl negative fibroblasts transfected with trk A or when these cells were exposed to gamma radiation. This result suggests that p53 preferentially binds to trk A in the presence of c-abl and that p53 and trk A do not appear to associate directly even if p53 is activated and its levels increased by gamma radiation. Overall, these data suggest that c-abl is possibly acting as an adaptor or bridge between p53 and trk A. Oncogene (2000).

Analysis of trk A and p53 association.

trk A tyrosine kinase (the high affinity receptor for nerve growth factor) binds to the p53 tumour suppressor protein in vitro and in vivo. Our aim was to determine which regions of p53 are involved in trk A association. In vitro binding experiments using baculovirus expressed trk A and in vitro transcribed and translated C-terminus p53 deletion mutants show amino acids 327-338 critical for association. Also, analysis with mutants at the N-terminus, conserved regions II, III, IV and V or amino acid positions 173, 175, 181, 248 and 249 (which are amino acids frequently mutated in a variety of neoplasms and transformed cell lines), show that these sites are not involved in trk A binding. Importantly, similar results are obtained after immunoprecipitation of lysates from p53 negative fibroblasts expressing trk A and the above p53 mutant proteins. These data suggest that the amino-terminus of the oligomerisation domain of p53 is involved in p53/trk A association.

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3.

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]-ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co-incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.