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We measured the effect(s) of CCR-5 genotype on disease progression by studying the frequency of a defective CCR-5 delta32 allele within a cohort of long-term infected individuals. An elevated frequency of CCR-5 delta32 heterozygotes within the cohort compared with a control population of blood donors was observed. An association between progression rate and CCR-5 delta32 heterozygosity was observed. Furthermore, analysis of proviral DNA V3 sequences from a subset of the cohort predicted that the majority of individuals (39 of 44) were infected with viruses predicted to utilize the beta-chemokine receptor CCR-5. The marked association between CCR-5 genotype and disease progression observed in this study may be a consequence of the predicted low frequency of CXCR-4-utilizing viruses present within the selected cohort.

We have previously shown that, during interleukin-2-driven G1/S transition in activated human T-lymphoblasts, a restricted group of cellular proteins become tyrosine phosphorylated de novo, and that p56lck is the major active tyrosine kinase at this stage of the cell cycle. We now report that okadaic acid, a potent and specific inhibitor of protein phosphatases type 1 and type 2A, inhibits S-phase entry, and that this occurs with the simultaneous disappearance of a 56 kDa tyrosine phosphoprotein. We show that this protein is the lck tyrosine kinase and that okadaic acid stimulates a mobility shift to 59 and 64 kDa forms. These two forms of lck were found to have decreased autocatalytic activity, as judged by immune-complex kinase assays. Two-dimensional phosphopeptide mapping and phosphoamino-acid analyses revealed that, in the presence of okadaic acid, lck becomes phosphorylated mainly on serine and to a lesser extent threonine, and that phosphorylation occurs at novel sites. These results show that the kinase activity of lck is at least partly regulated by protein phosphatases, and suggest a role for lck in directing growth-factor-mediated DNA synthesis during T-cell proliferation.

We report that in activated human T-lymphoblasts synchronized early in the G1-phase of the cell cycle, addition of interleukin 2 (IL-2) stimulates transition into S-phase in conjunction with specific and reproducible protein tyrosine phosphorylation events. Prominent among these was de novo phosphorylation of the p56lck tyrosine kinase, which appeared as a single polypeptide on SDS-polyacrylamide gel electrophoresis prior to the addition of IL-2, but as a doublet of approximately 56 and 59 kDa 3 h after IL-2 addition. Although the lck polypeptide doublet persisted into S-phase, the 59-kDa form was virtually undetectable in T-lymphoblasts in log phase growth. In T-lymphoblasts metabolically labeled with 32Pi, antiphosphotyrosine antisera identified a major 56-kDa tyrosine-phosphorylated protein, whereas antisera to lck showed that phosphorylation of lck occurred on both the 56- and 59-kDa forms. Phosphoamino acid analyses identified phosphoserine as the major phosphoamino acid on both of the lck polypeptides, although small amounts of phosphotyrosine were also detected on the 56-kDa form. Immune complex kinase assays revealed that only the lower band of the lck doublet exhibited autocatalytic activity. Furthermore, the 59-kDa form neither displayed autocatalytic activity, nor was it a substrate for phosphorylation by the 56-kDa form. We conclude that entry into S-phase in activated human T-lymphoblasts is associated with tyrosine phosphorylation of a limited number of proteins and suggest that p56lck activity is regulated during G1/S.

Treatment with metabolic inhibitors and addition of exogenous MgATP exerted different effects on secretion from streptolysin-O-permeabilized mast cells, responding to calcium and GTP-gamma-S as single effectors (i.e., independently of each other). These effects were also strongly dependent on the experimental conditions. Thus cells, triggered by Ca2+ at the time of permeabilization, did not require MgATP, but after metabolic inhibition rapidly became absolutely dependent on its provision, requiring high (> mM) concentrations. AMP-PNP was not effective. After longer treatment with metabolic inhibitors, the absolute dependence on MgATP was also exhibited by cells responding to dual effectors (i.e., Ca2+ and GTP-gamma-S applied together). In contrast, calcium independent secretion due to GTP-gamma-S was more resistant to metabolic inhibition, exhibiting no absolute requirements for MgATP. Once the responsiveness to GTP-gamma-S had been lost, it could not be restored by addition of MgATP. MgATP, in fact, inhibited the response of permeabilized cells to GTP-gamma-S. This effect could be mimicked by AMP-PNP. When permeabilized cells were washed before triggering, MgATP (0.1-1 mM concentration range) was no longer inhibitory but stimulatory. These differences between Ca(2+)- and GTP-gamma-S-induced responses indicate that ATP utilization is essential to the calcium, but not to the guanine nucleotide, pathway to secretion. The rate of the response to calcium/MgATP was much slower in the absence than in the presence of GTP-gamma-S. The onset of secretion occurred after an initial delay. This lag phase was abolished by addition of GTP-gamma-S, suggesting that a GTP-binding protein may control a reaction which constitutes a rate-limiting step in the secretory process.

The release of inflammatory mediators from mast cells occurs by a regulated exocytotic process. We have been able to study the intracellular events in this pathway by permeabilizing the plasma membrane of rat peritoneal mast cells and stimulating exocytosis by providing both Ca2+ and a guanine nucleotide. By this approach we have obtained evidence for the participation of at least two guanine nucleotide binding proteins in the control of exocytosis. We have also shown that ATP is unnecessary for the final events, but that it does have a number of modulatory functions, for instance in the control of the effective affinity of the proteins that bind Ca2+ and GTP. There is also evidence for a protein dephosphorylation in the later stages of the control pathway.

Mast cells permeabilized by streptolysin O secrete histamine and lysosomal enzymes in response to provision of a dual effector system comprising Ca2+ and a guanine nucleotide (e.g., GTP-gamma-S2) at concentrations in the micromolar range. These are both necessary and together they are sufficient. There is no requirement for adenosine triphosphate (ATP) and hence no obligatory phosphorylation reaction in the terminal stages of the exocytotic pathway. When exocytosis is induced by Ca2(+)-plus-GTP-gamma-S (i.e., no ATP) added at times after permeabilization (the permeabilization interval), cellular responsiveness declines so that there is no response to provision of the two effectors (both at 10(-5)M) if they are initially withheld and then added after 5 min. Here we show that this decline in responsiveness is characterized by a time-dependent reduction in the effective affinity for Ca2+. Affinity for Ca2+ and hence secretory competence can then be restored if ATP is added alongside the stimulus. Unlike cells stimulated to secrete at the time of permeabilization, exocytosis from cells that have undergone the cycle of permeabilization-induced refractoriness followed by ATP-induced restoration can be triggered by Ca2+ alone: after such conditioning there is no requirement for guanine nucleotide. In contrast, dependence on guanine nucleotide remains mandatory in cells that have been pretreated (i.e., before permeabilization) with okadaic acid (understood to be an inhibitor of protein phosphatases 1 and 2A) or phorbol myristate acetate (an activator of protein kinase C). These results indicate that obligatory dependence on guanine nucleotide is retained when the cells are treated under conditions conducive to maintained phosphorylation. It is concluded that the exocytotic mechanism of permeabilized mast cells is enabled by a dephosphorylation reaction and that the effector of the guanosine triphosphate (GTP)-binding protein (G epsilon) that mediates exocytosis is likely to be a protein phosphate.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.

Using rat mast cells permeabilized with streptolysin O we show that release of arachidonate generally occurs under similar but not identical conditions to those that cause exocytosis of beta-N-acetylglucosaminidase (hexosaminidase). Thus, hexosaminidase secretion and arachidonate release both require provision of Ca2+ together with a guanine nucleotide but exocytosis occurs at lower concentrations of both effectors. The kinetics of both processes are similar, with a delay in onset only when ATP is present. Arachidonate release occurs largely from a pool of arachidonyl phosphatidylcholine which appears to represent less than 1% of the total phosphatidylcholine of the cells. Despite the general similarity of the conditions causing exocytosis and arachidonate release, our results show that under some circumstances it is possible to obtain exocytosis without measurable release of arachidonate and that therefore phospholipase A2 activation is not an essential precursor of secretion.

Recent advances in the understanding of the terminal stages of the pathway of regulated secretion (exocytosis) have depended in large part on the use of permeabilized secretory cells in which the cytosol is directly accessible to manipulation from the outside. As well as Ca2+, a role for GTP, and hence a GTP-binding protein (GE), is plainly apparent in the exocytotic mechanism of some cells. In metabolically depleted and permeabilized mast cells, the combination of Ca2+ and a guanine nucleotide are a sufficient stimulus for the release of the total cell content of histamine and hexosaminidase. ATP is not required and so a phosphorylation reaction does not comprise a necessary step in the terminal pathway. Phosphorylating nucleotides, however, can modulate the exocytotic reaction in a manner that suggests that the reverse reaction, namely protein dephosphorylation, might be an enabling step. Support for this idea is derived from experiments in which the cells are conditioned to become responsive to Ca2+ alone; in these circumstances the dependence of secretion on the presence of a guanine nucleotide can be reimposed by okadaic acid, an inhibitor of protein phosphatases.