Modulation of Mcl-1 by BLyS, rapamycin and Pim; reduction of peripheral B-cell populations in Pim 1 and 2 doubly deficient mice receiving rapamycin in vivo; and a minimal molecular model of BLyS-dependent growth and survival. (A) Modulation of Mcl-1 protein by BLyS, rapamycin, and Pim. Extracts of small resting wild-type or Pim 1^−/−2^−/− B cells either freshly isolated (*) or cultured for 2 days in CM with or without rhuBLyS (50 ng/mL) and with vehicle or rapamycin (50 nM) were electrophoretically separated and transferred to nitrocellulose membranes. Membranes were probed with anti–Mcl-1 antibody, stripped, and reprobed with anti-actin antibody. (B) Peripheral B-cell survival in rapamycin-treated mice; 5- to 8-week-old Pim 1^−/−2^−/− doubly deficient or normal littermates were injected intraperitoneally with 2.5 mg/kg of rapamycin or diluent every other day for 6 days and killed on day 7. The number of viable spleen cells from individual mice was determined by counting and trypan blue exclusion. Peripheral B-cell subpopulations were identified by flow cytometry of viable lymphocytes stained for B220, CD 21/35 and CD23. Total B lymphocytes (B220⁺; □ represents rapamycin-treated donors; ■, diluent-treated donors) were subdivided into marginal zone (MZ: B220⁺, CD21/35^hi, and CD23^lo; ◇ represents rapamycin-treated donors; ♦, diluent-treated donors) or follicular (Fo: B220⁺, CD21/35^int, CD23^hi; △ represents rapamycin-treated donors; ▲, diluent-treated donors) B-cell subpopulations, and their number in each donor spleen was determined by reference to the splenocyte counts. Data pooled from 2 independent experiments are shown. (C) BLyS-mediated signaling pathways regulating B-cell growth and survival. Stimulation of B cells by BLyS leads to the activation of PI3-kinase, which phosphorylates Akt on T308⁴²  (current study) and by a separate pathway, the processing and nuclear localization of NF-κB2 with the subsequent induction of pim 2 mRNA and production of constitutively active Pim 2 protein.^15,17 ,34  (current study). Akt is fully activated by phosphorylation on S473 mediated by the rictor containing TOR complex mTORC2³⁰  (current study). Akt and the raptor containing TOR complex mTORC1 converge to promote rapamycin-sensitive cell growth^31,32  (current study) by inducing glucose uptake and use and up-regulating amino acid and iron transporters, which maintain cell size and promote atrophy resistance^28,31,47  (current study). Pim 2 also increases glycolysis and promotes maintenance of cell size by a rapamycin-independent, glucose-dependent mechanism^34,35  (current study). BLyS-induced survival requires the antiapoptotic Bcl-2 family member Mcl-1²²  (current study). Akt induces the activation of CREB and NF-κB1,⁴⁴  which promote mcl-1 transcription.^73,74  Akt also inactivates FOXO3a,^48,56  (current study), suppressing the transcription of the proapoptotic protein Bim, a major regulator of B-cell homeostasis.^55,57  Mcl-1 protein levels are controlled at the level of translation by mTORC1 and Pim 2, which inactivate the translational repressor 4E-BP1 by rapamycin-dependent and -independent processes; Mcl-1 protein is stabilized by Erk,⁷⁶  reported to be induced by BLyS stimulation,²⁰  whereas Mcl-1 turnover is enhanced by the action of Gsk-3b,⁷⁵  which is inhibited by active Akt⁴⁸  (current study). Mcl-1 protein is reduced by rapamycin treatment⁵⁴  (current study). Reference citations in this legend for activities induced by BLyS stimulation are in bold. AA indicates amino acid.