Biomedical subjects

Galit Shohat

Publications and source records attributed to Galit Shohat.

6 recordsLinked to original sources

PubMed2006

Hsp90 recognizes a common surface on client kinases.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Amino Acid Sequence↗

PubMed2004

Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions.

The death-associated protein (DAP) kinase family includes three protein kinases, DAP kinase, DAP kinase-related protein 1, and ZIP kinase, which display 80% amino acid identity within their catalytic domains and are functionally linked to common subcellular changes occurring during cell death, such as the process of membrane blebbing. Here we show physical and functional cross talk between DAP kinase and ZIP kinase. The two kinases display strong synergistic effects on cell death when coexpressed and physically bind each other via their catalytic domains. Furthermore, DAP kinase phosphorylates ZIP kinase at six specific sites within its extracatalytic C-terminal domain. ZIP kinase localizes to both the nucleus and the cytoplasm and fractionates as monomeric and trimeric forms. Significantly, modification of the DAP kinase phosphorylation sites influences both the localization and oligomerization status of ZIP kinase. A mutant ZIP kinase construct, in which the six serine/threonine residues were mutated to aspartic acid to mimic the phosphorylated state, was found predominantly in the cytoplasm as a trimer and possessed greater cell death-inducing potency. This suggests that DAP kinase and ZIP kinase function in a biochemical pathway in which DAP kinase activates the cellular function of ZIP kinase through phosphorylation, leading to amplification of death-promoting signals.

Apoptosis↗

PubMed2003

Protein tyrosine phosphatase epsilon inhibits signaling by mitogen-activated protein kinases.

Mitogen-activated protein kinases (MAPKs) mediate signaling from the cell membrane to the nucleus following their phosphorylation at conserved threonine and tyrosine residues within their activation loops. We show that protein tyrosine phosphatase epsilon (PTP epsilon) inhibits ERK1 and ERK2 kinase activity and reduces their phosphorylation; in agreement, ERK phosphorylation is increased in fibroblasts and in mammary tumor cells from mice genetically lacking PTP epsilon. PTP epsilon inhibits events downstream of ERKs, such as transcriptional activation mediated by Elk1 or by the serum response element. PTP epsilon also inhibits transcriptional activation mediated by c-Jun and C/EBP binding protein (CHOP) but not that mediated by the unrelated NFkB, attesting that it is broadly active within the MAPK family but otherwise specific. The effect of PTP epsilon on ERKs is at least in part indirect because phosphorylation of the threonine residue in the ERK activation loop is reduced in the presence of PTP epsilon. Nonetheless, PTP epsilon is present in a molecular complex with ERK, providing PTP epsilon with opportunity to act on ERK proteins also directly. We conclude that PTP epsilon is a physiological inhibitor of ERK signaling. Slow induction of PTP epsilon and its lack of nuclear translocation following mitogenic stimulation suggest that PTP epsilon functions to prevent inappropriate activation and to terminate prolonged, rather than acute, activation of ERK in the cytosol.

3T3 Cells↗

PubMed2002

The DAP-kinase family of proteins: study of a novel group of calcium-regulated death-promoting kinases.

DAP-kinase (DAPk) is a Ca(2+)/calmodulin (CaM)-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It associates with actin microfilament and has a unique multidomain structure. One of the substrates of DAPk was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Four additional kinases have been identified based on the high homology of their catalytic domain to that of DAPk. Yet, they differ in the structure of their extracatalytic domains and in their intracellular localization. One member of this family, DRP-1, also shares with DAPk both the property of activation by Ca(2+)/CaM and a specific phosphorylation-based regulatory mechanism. The latter involves an inhibitory type of autophosphorylation on a conserved serine at position 308, in the CaM regulatory domains of these two kinases. This phosphorylation, which occurs in growing cells, restrains the death-promoting effects of these kinases, and is specifically removed upon exposure of cells to various apoptotic stimuli. The dephosphorylation at this site increases the binding and sensitivity of each of these two kinases to their common activator-CaM. In DAPk, the dephosphorylation of serine 308 also increases the Ca(2+)/CaM-independent substrate phosphorylation. In DPR-1, it also promotes the formation of homodimers necessary for its full activity. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. In DRP-1, it introduces an additional locking device by preventing homodimerization. We propose that this unique mechanism of autoinhibition, evolved to keep these death-promoting kinases silent in healthy cells and ensures their activation only in response to apoptotic signals.

Amino Acid Sequence↗

PubMed2002

The caspase-cleaved DAP5 protein supports internal ribosome entry site-mediated translation of death proteins.

Apoptosis is characterized by a translation switch from cap-dependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and death-associated protein 5 (DAP5) proteins (eIF4GI M-FAG/p76 and DAP5/p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5/p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG/p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5/p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.

Apoptosis↗

PubMedDate not supplied

The regulation of death-associated protein (DAP) kinase in apoptosis.

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Amino Acid Substitution↗