BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on posttranslational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events -mediated through separate signaling processes and involving different target sites -in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERKinducible versus ERK-independent sites.

Protein kinase C activators and microtubule-damaging drugs stimulate BCL2 phosphorylation, which has been associated with either enhancement or inhibition of cell viability. In a Burkitt lymphoma cell line, both types of agents likewise stimulated phosphorylation of myeloid cell leukemia 1 (MCL1), another viability-promoting BCL2 family member. However, while MCL1 phosphorylation induced by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not affect its electrophoretic mobility, microtubuledamaging agents, such as taxol, induced MCL1 phosphorylation associated with a band shift to decreased mobility. Inhibitors of extracellular signal-regulated kinase (ERK) activation blocked TPA-induced MCL1 phosphorylation but not the taxol-induced band shift. TPA-induced MCL1 phosphorylation occurred rapidly and was not associated with decreased viability, while the taxol-induced band shift occurred upon extended exposure as cells accumulated in G 2 /M followed by cell death. Protein phosphatase 1/2A inhibitors also induced the MCL1 band shift/phosphorylation. Thus, MCL1 undergoes two distinct types of phosphorylation: (i) TPAinduced, ERK-associated phosphorylation, which does not alter the electrophoretic mobility of MCL1, and (ii) ERK-independent phosphorylation, which results in an MCL1 band shift and is induced by events in G 2 /M or protein phosphatase 1/2A inhibitors.Both anti-and proapoptotic BCL2 family members undergo phosphorylation, as is seen with BCL2, BCLX, and BAD. The role of this post-translational modification has been well defined for the proapoptotic family member, BAD, where phosphorylation promotes association with a 14-3-3 protein instead of BCLX, thereby freeing BCLX to exert its antiapoptotic activity (1-6). However, the role of phosphorylation in the case of BCL2 and other antiapoptotic family members is not yet completely understood. BCL2 phosphorylation is induced by several types of agents. These include growth factors and protein kinase C activators, such as erythropoietin, interleukin-3, and bryostatin-1 (7-9). They also include microtubule-directed agents, such as taxol and nocodazole, as well as the protein phosphatase 1/2A inhibitor, okadaic acid (10 -18). BCL2 phosphorylation has been associated with viability promotion in some cases and with cell death in others (8, 18). These varying results could relate to the fact that BCL2 phosphorylation has been studied by several investigators using different agents in varied cell lines.Our studies focus on myeloid cell leukemia 1 (MCL1), 1 an antiapoptotic BCL2 family member identified by its rapid up-regulation during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of myeloid ML-1 cells (19). Previous studies on MCL1 regulation indicated that TPA increases MCL1 expression within 3 h through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signal transduction pathway, which activates a serum response factor-Elk-1 transcription factor complex (20, 21). Microtubule-disrupting agents such...

The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine) and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA)-induced activation of extracellular signal-regulated kinase (ERK), is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3)-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an ∼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance – effects that can be suppressed by inhibition of ERK activation.

The antiapoptotic BCL2 family member MCL1 is normally upand down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK-and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G 2 /M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK-and GSK3 (but not G 2 /M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death.The antiapoptotic mediator MCL1 was discovered based on rapid, transient up-regulation in ML-1 human myeloblastic leukemia cells initiating differentiation in the presence of 12-Otetradecanoylphorbol 13-acetate (TPA) 7 (1). MCL1 has since been found to be induced in cells at various stages in differentiation, in response to specific growth, differentiation, and survival factors. Induction of MCL1 expression serves to promote viability in cells undergoing changes in proliferation or differentiation or responding to stress, infection, or other signals. Thus, when MCL1 is conditionally knocked out, hematopoietic stem cells do not survive, and early stage B-or T-lymphocytes die instead of continuing their differentiation (2, 3); mature lymphoid cells likewise exhibit impaired survival despite the presence of growth factors (4, 5). Just as induction of MCL1 expression serves to promote viability, its down-regulation is an early, pivotal event in apoptosis initiated by DNA damage and other death stimuli (6, 7). Overall, in response to changing signals, increases and decreases in MCL1 expression regulate cell viability, maintaining cells and lineages that are needed while eliminating those that are no longer needed or are damaged or overabundant (1).Although MCL1 is normally expressed in particular cells in response to specific sti...

The E600A mutation in the NOD2 gene may confer a higher penetrance of uveitis but a later onset of milder forms of non-ocular involvement.