The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PPl) and 2A (PPZA) with K, values below 0.1 nM. Protein phosphatase 2B is inhibited lOOO-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PPZA, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PPI. We discuss the possibility that inhibition of PPl and PPZA accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.

The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1-2 nM okadaic acid. In contrast, type l protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 #M okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.

Despite the characterization of neuroprotection by transforming growth factor-beta1 (TGF-beta1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-beta1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-beta1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser(112) in mouse brains after adenovirus-mediated TGF-beta1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-beta1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-beta1 inhibited the increase in Bad expression caused by staurosporine. TGF-beta1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-beta1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-beta1 in cultured hippocampal cells. Together, we demonstrate that TGF-beta1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

Protein phosphorylation is a key mechanism for intracellular signal transduction in both prokaryotic and eukaryotic cells. Vertebrate proteins are prevalently phosphorylated on side chains that contain a hydroxyl group, such as serine, threonine and tyrosine residues. In the past decade, however, an increasing number of examples of histidine phosphorylation has been described. Because acid treatment of phosphoproteins during purification and detection of phosphoamino acid analysis is routine, O-phosphomonoesters have been studied more often, and the existence of acid-labile phosphates has been largely overlooked. The latter class of N-phosphoamidates may well be more widespread than is generally believed, even though the O-phosphates remain the major class in terms of quantity and extent of distribution in proteins. Phosphohistidine currently is estimated to be 10- to 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine [Matthews, H.R. (1995) Pharmac. Ther. 67, 323-350.]. This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria. It also describes the still limited, yet increasing, data from homologs of the bacterial two-component system. Finally, novel mechanisms of phosphorylation and dephosphorylation of histidine residues not related to the two-component system are described.

No abstract