The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein

Pleckstrin is a modular platelet protein consisting of N- and C-terminal pleckstrin homology (PH) domains, a central dishevelled egl10 and pleckstrin (DEP) domain and a phosphorylation region. Following agonist-induced platelet stimulation, dimeric pleckstrin translocates to the plasma membrane, is phosphorylated and then monomerizes. A recent study found that pleckstrin null platelets from a knockout mouse have a defect in granule secretion, actin polymerization and aggregation. However, the mechanism of pleckstrin signaling for this function is unknown. Our recent studies have led to the identification of a novel pleckstrin-binding protein, serum deprivation response protein (SDPR), by co-immunoprecipitation, GST-pulldowns and nanospray quadruple time of flight mass spectrometry. We show that this interaction occurs directly through N-terminal sequences of pleckstrin. Both pleckstrin and SDPR are phosphorylated by protein kinase C (PKC), but the interaction between pleckstrin and SDPR was shown to be independent of PKC inhibition or activation. These results suggest that SDPR may facilitate the translocation of nonphosphorylated pleckstrin to the plasma membrane in conjunction with phosphoinositides that bind to the C-terminal PH domain. After binding of pleckstrin to the plasma membrane, its phosphorylation by PKC exerts downstream effects on platelet aggregation/secretion.

WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.