Protein Kinase D Is a Positive Regulator of Bit1 Apoptotic Function

Abstract

Bit1 (Bcl-2 inhibitor of transcription) is a mitochondrial protein that induces caspase-independent apoptosis upon its release into the cytoplasm. Bit1 is primarily associated with anoikis (cell death induced by detachment from the extracellular matrix), because the apoptotic function of Bit1 is inhibited by integrin-mediated cell attachment but not by many other antiapoptotic treatments. Here, we show that protein kinase D (PKD) regulates Bit1 apoptotic function. Overexpression of constitutively active PKD or PKD activation by treatment with phorbol 12-myristate 13-acetate results in phosphorylation of two serine residues (Ser 5 and Ser 87 ) in a form of Bit1 that is confined to the cytoplasm and concomitantly increases the apoptotic activity of cytoplasmic Bit1. Conversely, suppressing PKD activity with pharmacological inhibitors or small interfering RNA approaches attenuates apoptosis induced by cytoplasmic Bit1. Furthermore, PKD regulates induction of cell death by wild-type Bit1 following loss of cell attachment to the extracellular matrix. Activation of PKD enhances Bit1 function in anoikis, whereas inhibiting PKD function with pharmacological inhibitors or small interfering RNA compromises the ability of Bit1 to induce anoikis. The induction of Bit1-mediated apoptosis by PKD is in part attributable to the release of Bit1 from mitochondria to the cytoplasm as a consequence of phosphorylation of Ser 5 in the mitochondrial localization sequence of Bit1. Consistent with the regulatory role of PKD in the anoikis function of Bit1, we found that cell attachment to fibronectin inhibits PKD activity. These studies identify the PKD serine/threonine kinase as one of the signaling molecules through which integrin-mediated cell attachment controls Bit1 activity and anoikis.

Bit-1 Mediates Integrin-dependent Cell Survival through Activation of the NFκB Pathway

“…When cells lose their attachment to the underlying ECM and no integrin is ligated, Bit-1 is released from mitochondria into the cytoplasm where it binds to the proapoptotic AES-TLE complex and enhances anoikis (11). This may be due to phosphorylation of Bit-1 by other signaling molecules such as PKD1 (13). However in cells attached to fibronectin, Bit-1 works in conjunction with a FAK/PI3K/NFB signaling pathway and elevates bcl-2 transcription to promote cell survival.…”

“…Interestingly, Bit-1 and FAK also associated on collagen IV, suggesting that the signaling complex requires more than the physical association. Bit-1 phosphorylation by other proteins also may be involved (13). Integrin attachment promotes cell survival through the PI3K pathway via FAK, and both ␣5␤1 and ␣v␤3 pro-survival signals are through PI3K (3).…”

“…In this way, Bit-1 acts as a downstream activator of anoikis. Recently, the serine/threonine kinase PKD1 was shown to phosphorylate Bit-1 on two serines (Ser 5 and Ser 87 ) in a mutant form of Bit-1 that is confined to the cytoplasm (13), suggesting that phosphorylation of Bit-1 may affect its function. Overexpression of Bit-1 was also found to impair Erk phosphorylation (14).…”

Pathological implication and function of Bcl2-inhibitor of transcription in ovarian serous papillary adenocarcinomas

“…Griffiths and colleagues demonstrated that the NF-κB pathway plays an important role in Bit1-mediated cell survival [14]. Hector Biliran et.al reported that protein kinase 1(PKD) is a positive regulator of Bit1 apoptotic functions [15], while according to Rania Kairouz-Wahbe, Bit1 can negatively regulate Erk activity [16]. Such investigations suggest that Bit1-induced anoikis is controlled by sophisticated regulation network, and is closely associated with other pathways and cell activities.…”

Regulation of Protein Kinase D1 Activity

“…The observations that PKD1-Ser 910 phosphorylation increases in the context of PKD1 activation by growth factor receptors or phorbol esters and that constitutively active forms of PKD1 (such as the PH domain-deleted or S738E/S742E-substituted mutants) display high levels of basal Ser 910 phosphorylation led to the widespread use of PKD1-Ser 910 phosphorylation as a surrogate marker of A, G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) activate PKD1 via an allosteric mechanism involving lipid cofactors and phosphorylation by nPKC isoforms. PKD1 then phosphorylates a range of cellular substrates, including HDAC5, the sarcomeric proteins cTnI and cardiac myosin binding protein-C (cMyBP-C), CREB, the 27-kDa heat shock protein (HSP27), p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4), cJun, Bit1 (Bcl-2 inhibitor of transcription, a mitochondrial protein that induces caspase-independent apoptosis), the F-actin-binding protein cortactin, the cofillin phosphatase slingshot 1, RIN1 (a Ras effector protein that influences ERK and c-Abl pathways), and the p85 regulatory subunit of PI3K (which is inhibited-no longer binds to RTKs-when phosphorylated in the SH2 domain by PKD1); direct substrates of PKD1 are in pink (Hurd et al, 2002;Döppler et al, 2005;Biliran et al, 2008;Eiseler et al, 2009Eiseler et al, , 2010Peterburs et al, 2009;Barišić et al, 2011;Lee et al, 2011;Spratley et al, 2011;Ziegler et al, 2011). B and C depict alternative mechanism for PKD1 regulation by reactive oxygen species (ROS) or caspase-3 in the setting of oxidative stress or apoptosis (see Other PKD1 Activation Mechanisms).…”