Pramod Pandey

Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90

The release of cytochrome c from mitochondria results in the formation of an Apaf‐1–caspase‐9 apoptosome and induces the apoptotic protease cascade by activation of procaspase‐3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf‐1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf‐1 and thereby inhibits cytochrome c‐mediated activation of caspase‐9. Addition of purified Apaf‐1 to Hsp90‐depleted cytosolic extracts restores cytochrome c‐mediated activation of procaspase‐9. We also show that Hsp90 inhibits cytochrome c‐mediated oligomerization of Apaf‐1 and thereby activation of procaspase‐9. Furthermore, treatment of cells with diverse DNA‐damaging agents dissociates the Hsp90–Apaf‐1 complex and relieves the inhibition of procaspase‐9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c‐dependent apoptosis and for involvement of a chaperone in the caspase cascade.

Translocation of SAPK/JNK to mitochondria and interaction with Bcl-xL in response to DNA damage.

Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis. We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association of SAPK with the anti-apoptotic Bcl-xL protein. SAPK phosphorylates Bcl-xL on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-xL, a mutant Bcl-xL with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with Bcl-xL in the apoptotic response to genotoxic stress.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no effect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These findings define a novel function for Hsp27 and provide the first evidence that a heat shock protein represses cas-3 activation.

Mitochondrial translocation of protein kinase C delta in phorbolester-induced cytochrome C release and apoptosis

Apoptosis is induced by the release of cytochromec from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) δ from the cytoplasm to mitochondria. The results also show that translocation of PKCδ results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCδ translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCδ to mitochondria is responsible, at least in part, for inducing cytochrome crelease and apoptosis.

Inactivation of DNA-Dependent Protein Kinase by Protein Kinase Cδ: Implications for Apoptosis

ABSTRACT Protein kinase Cδ (PKCδ) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCδ in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCδ (PKCδ CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCδ cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCδ associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCδ CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCδ CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCδ CF. These findings support the hypothesis that functional interactions between PKCδ and DNA-PK contribute to DNA damage-induced apoptosis.

Dexamethasone induces apoptosis of multiple myeloma cells in a JNK/SAP kinase independent mechanism

The stress-activated protein kinases (SAPKs), also known as c-Jun amino-terminal kinases (JNKs), are activated in response to diverse stimuli including DNA damage, heat shock, interleukin-1, tumor necrosis factor-α and Fas. Although all these inducers cause apoptosis, whether SAPK/JNK activation is required for apoptosis is controversial. In this study, we demonstrate that ionizing radiation (IR) and dexamethasone (Dex) induce apoptosis in multiple myeloma (MM) derived cell lines, as well as in patient cells. IR-induced apoptosis is associated with activation of SAPK/JNK and p38 kinase, in contrast to Dex-induced apoptosis, which is not associated with activation of stress kinases. Moreover, Dex-induced apoptosis is associated with a significant decrease in the activities of mitogen activated protein kinase (MAPK) and p70S6K, whereas IR-treatment does not alter the activity of these kinases. Both IR and Dex induce poly (ADP ribose) polymerase (PARP) cleavage, a signature event of apoptosis. Finally, interleukin-6 (IL-6) inhibits Dex-induced apoptosis, downregulation of MAP and p70S6K growth kinases and PARP cleavage; in contrast, IL-6 does not inhibit IR-induced apoptosis, activation of SAPK/JNK, and PARP cleavage. Taken together, our findings suggest that SAPK/JNK activation is not required for apoptosis in MM cells, and that there are at least two distinct apoptotic signaling pathways: (i) SAPK/JNK-associated, which is induced by IR and unaffected by IL-6; and (ii) SAPK/JNK-independent, which is induced by Dex, associated with downregulation of MAPK and p70S6K and inhibited by IL-6.

Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase

BACKGROUND Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. RESULTS We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. CONCLUSIONS The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

Activation of p38 Mitogen-activated Protein Kinase by PYK2/Related Adhesion Focal Tyrosine Kinase-dependent Mechanism

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.

Targeting of the c-Abl Tyrosine Kinase to Mitochondria in Endoplasmic Reticulum Stress-Induced Apoptosis

ABSTRACT The ubiquitously expressed c-Abl tyrosine kinase localizes to the nucleus and cytoplasm. Using confocal microscopy, we demonstrated that c-Abl colocalizes with the endoplasmic reticulum (ER)-associated protein grp78. Expression of c-Abl in the ER was confirmed by immunoelectron microscopy. Subcellular fractionation studies further indicate that over 20% of cellular c-Abl is detectable in the ER. The results also demonstrate that induction of ER stress with calcium ionophore A23187, brefeldin A, or tunicamycin is associated with translocation of ER-associated c-Abl to mitochondria. In concert with targeting of c-Abl to mitochondria, cytochrome c is released in the response to ER stress by a c-Abl-dependent mechanism, and ER stress-induced apoptosis is attenuated in c-Abl-deficient cells. These findings indicate that c-Abl is involved in signaling from the ER to mitochondria and thereby the apoptotic response to ER stress.

RAFTK/PYK2-dependent and -independent apoptosis in multiple myeloma cells

Related Adhesion Focal Tyrosine Kinase (RAFTK; also known as Pyk2), is a member of the Focal Adhesion Kinase (FAK) subfamily and is activated by TNFα, UV light and increases in intracellular calcium levels. However, the function of RAFTK remains largely unknown. Our previous studies demonstrated that treatment with dexamethasone (Dex), ionizing radiation (IR), and anti-Fas mAb induces apoptosis in multiple myeloma (MM) cells. In the present study, we examined the potential role of RAFTK during induction of apoptosis in human MM cells triggered by these three stimuli. Dex-induced apoptosis, in contrast to apoptosis triggered by anti-Fas mAb or IR, is associated with activation of RAFTK. Transient overexpression of RAFTK wild type (RAFTK WT) induces apoptosis, whereas transient overexpression of Kinase inactive RAFTK (RAFTK K-M) blocks Dex-induced apoptosis. In contrast, transient overexpression of RAFTK K-M has no effect on apoptosis triggered by IR or Fas. In Dex-resistant cells, Dex does not trigger either RAFTK activation or apoptosis. Finally, interleukin-6 (IL-6), a known survival factor for MM cells, inhibits both activation of RAFTK and apoptosis of MM.1S cells triggered by Dex. Our studies therefore demonstrate Dex-induced RAFTK-dependent, and IR or Fas induced RAFTK-independent apoptotic signaling cascades in MM cells.