Insights into the importance of hydrogen bonding in the gamma-phosphate binding pocket of myosin: structural and functional studies of serine 236.

The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during gamma-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP.vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A.MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values.

Activation of maxi-anion channel by protein tyrosine dephosphorylation.

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)zeta, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPzeta gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPzeta in mouse fibroblasts, but not in C127 cells.

Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70.

Heat shock proteins of the hsp/hsc70 family are essential chaperones, implicated in the stress response, aging, and a growing number of human diseases. At the molecular level, hsc70s are required for the proper folding and intracellular targeting of polypeptides as well as the regulation of apoptosis. Cytoplasmic members of the hsp/hsc70 family are believed to shuttle between nuclei and cytoplasm; they are found in both compartments of unstressed cells. Our experiments demonstrate that actin filament-destabilizing drugs trigger the nuclear accumulation of hsc70s in unstressed and heat-shocked cells recovering from stress. Using human-mouse heterokaryons, we show that stress inhibits shuttling and sequesters the chaperone in nuclei. The inhibition of hsc70 shuttling upon heat shock is only transient, and transport is reestablished when cells recover from stress. Hsc70 shuttling is controlled by hsc70 retention in the nucleus, a process that is mediated by two distinct mechanisms, ATP-sensitive binding of hsc70s to chaperone substrates and, furthermore, the association with nucleoli. The nucleolar protein fibrillarin and ribosomal protein rpS6 were identified as components that show an increased association with hsc70s in the nucleus upon stress exposure. Together, our data suggest that stress abolishes the exit of hsc70s from the nucleus to the cytoplasm, thereby limiting their function to the nuclear compartment. We propose that during recovery from stress hsc70s are released from nuclear and nucleolar anchors, which is a prerequisite to restore shuttling.

Regulation of M-type potassium current by intracellular nucleotide phosphates.

The effects of intracellular application of various concentrations of adenine nucleoside phosphates and nucleotide analogs on the M-type K current (IM) of single neurons isolated from sympathetic ganglia were studied. With 1 mM MgATP intracellularly IM decreased to 25% of its initial level 39 min after the start of whole-cell recording. In the absence of ATP the current decreased more rapidly. Addition of glucose and pyruvate extracellularly was equivalent to adding 1 mM MgATP intracellularly. AMP-PNP, a nonhydrolyzable ATP analog, at a concentration of 1 or 3 mM was unable to maintain IM in the absence of ATP. When ATP and AMP-PNP were combined in the pipette, however, the maintenance of IM was prolonged. A series of nucleotides and analogs have been combined with ATP to test for their ability to maintain IM and to alter calcineurin phosphatase activity. There was a positive correlation between the ability of a nucleotide to prevent the rundown of IM and its ability to inhibit calcineurin phosphatase activity. These findings show that the amplitude of IM is dually regulated by cellular levels of adenine nucleotide diphosphates and triphosphates. A hydrolyzable form of ATP is necessary to maintain the M current. The maintenance of IM is further enhanced by the simultaneous presence of ADP or other adenine nucleotides that alter calcineurin activity, but not by higher concentrations of ATP alone. These results are consistent with regulation of IM by phosphorylation events that maintain IM and dephosphorylation events that lead to current rundown.