Adelina A. Davies

The gene coding for the p68 calcium-binding protein is localised to bands q32–q34 of human chromosome 5, and to mouse chromosome 11

SummaryThe gene coding for human p68, a membrane-associated calcium-binding protein, has been assigned to chromosome 5, using a cDNA clone to probe genomic DNA from rodent-human somatic cell hybrids by Southern hybridisation. The gene was localised, by in situ hybridisation, to 5q32–34. The murine gene was assigned to chromosome 11, using a murine cDNA clone to probe genomic DNA from rodent-rodent somatic cell hybrids.

A growth-dependent post-translational modification of annexin VI

Annexin VI (p68, 67-kDa calelectrin) is a member of a family of Ca2+/phospholipid-binding proteins, that includes p35 (annexin I) and p36 (annexin II), the major cellular substrates for phosphorylation by the epidermal growth factor receptor and pp60v-src tyrosine kinase activities, respectively. We report here that like annexins I and II, annexin VI is phosphorylated in vivo, but that in contrast, annexin VI phosphorylation is associated with cell growth. In both Swiss 3T3 fibroblasts and human T-lymphoblasts the pattern of phosphorylation followed an almost identical profile. In particular, annexin VI was not phosphorylated in quiescent cells, but was phosphorylated on serine and to a lesser extent threonine, several hours following cell stimulation. Furthermore, annexin VI also incorporated phosphate in a growth-dependent manner, in a form other than a phosphoamino-acid. The phosphate was visualised following acid hydrolysis of immunoprecipitated annexin VI, as part of a complex having high mobility on 2-D thin-layer electrophoresis. The identity of this complex is not known. The results suggest that a post-translational modification other than direct protein phosphorylation may influence the activity of annexin VI and provide evidence linking cell growth with regulation of annexin VI function.

Activation of protein kinase C modulates the expression of the T3/T cell antigen receptor complex on human T lymphocytes

Activators of protein kinase C induced a rapid decrease (within 15 min) in the surface expression of the T3 antigen and T-lymphocyte antigen receptor (Ti) on HPB-ALL cells, and a concomitant phosphorylation of the T3 γ and δ polypeptides; the γ chain was more extensively phosphorylated than the δ chain. No phosphorylation of the T3 ∈ chain and the Ti α and β polypeptides was detected. Evidence was obtained that the T3 γ chain is phosphorylated only on serine residues.

Comparative analysis of phosphotyrosyl polypeptides in normal and leukemic human T lymphocytes activated via CD3 or CD2

Phosphotyrosyl polypeptides induced following CD3- or CD2- specific antibody stimulation were analysed in different human T cell lines by immunoblotting or by immunoprecipitation of 32P-labelled cell lysates using a phosphotyrosine-specific monoclonal antibody. In Jurkat cells, resting peripheral T lymphocytes, T lymphoblasts, CD8+ T lymphoblasts and a CD4+ T cell clone, CD3 stimulation induced a strong but transient tyrosine phosphorylation of at least 15 polypeptides. However, in peripheral T cells and T blasts, the kinetics of phosphorylation were considerably slower than in Jurkat cells. The pattern of phosphotyrosyl polypeptides induced by CD3 stimulation was similar, although some differences were noted between normal T cells and Jurkat, especially at the level of the extent of phosphorylation. As had been previously reported for Jurkat T cells, a qualitatively similar tyrosine phosphorylation response was induced upon CD2 or CD3 stimulation in each of the analysed T cell populations, suggesting that CD3 and CD2 share a common pathway of protein tyrosine kinase (PTK) activation. In HPB. ALL leukemia T cells (which express very low levels of CD45), both CD3 and CD2 stimulation induced only very weak protein tyrosyl phosphorylation. However, a 50 kDa polypeptide, which was part of an inducible doublet in Jurkat or normal T lymphocytes, was constitutively tyrosyl-phosphorylated in the HPB. ALL line. These results suggest that there is a common pathway of early PTK activation following CD3- or CD2-mediated stimulation in mature T cells, whether they express surface CD4 or CD8, and also that the PTK may be differently regulated in different T cell populations leading to different kinetics or intensity of tyrosyl phosphorylation.

Ca2+-Binding Proteins Located on the Cytoplasmic Face of the Lymphocyte Plasma Membrane

Three polypeptides of mol. wts.~68,000, 33,000 and 28,000 have been identified in Ca2+ -chelator extracts of purified preparations of lymphocyte plasma membrane (p.m.). They are preferentially associated with the actin-rich, Nonidet P40-insoluble fraction of the membrane and are not labelled by lactoperoxidase-catalyzed iodination of whole lymphocytes, suggesting that they are located on the cytoplasmic face of the p.m. The 68,000 mol. wt. protein, the most abundant of the three in the EGTA extract, has been purified from pig and human lymphocyte p.m. and partially characterized. It is a monomeric acidic protein with a single high-affinity Ca2+-binding site (KD = 1.2 μM). From their solubility with Ca2+ -chelators present, the 33,000 and 28,000 components seem to be Ca2+-binding proteins too.