Cutting Edge: Differential Signaling Requirements for Activation of Assembled D3-cdk4 Complexes in B-1 and B-2 Lymphocyte Subsets This information is current as of October 2, 2021. Debra A. Tanguay, Thomas P. Colarusso, Cheryl Doughty, Sandra Pavlovic-Ewers, Thomas L. Rothstein and Thomas C. Chiles J Immunol 2001; 166:4273-4277; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Differential Signaling Requirements for Activation of Assembled Cyclin D3-cdk4 Complexes in B-1 and B-2 Lymphocyte Subsets1

Debra A. Tanguay,2* Thomas P. Colarusso,3†§ Cheryl Doughty,* Sandra Pavlovic-Ewers,4* Thomas L. Rothstein,†‡§ and Thomas C. Chiles5*

to the peritoneum, whereas B-2 cells predominate in spleen and Downloaded from B-1 lymphocytes represent a distinct B cell subset with unusual lymph nodes. B-1 cells contribute substantial proportions of non- mitogenic responses. PMA alone promotes proliferation in B-1 immune (resting) IgM and IgA that is repertoire restricted. cells, but not in splenic B-2 cells. Although -cyclin- Whether B-1 cells represent a developmental lineage distinct from dependent kinase 4 (cdk4) complexes mediate early retinoblas- B-2 cells or, alternatively, derive from a single B cell lineage in toma product (pRb) phosphorylation in B-1 cells, the which B-2 cells differentiate to B-1 cells in response to B cell Ag transient nature of their accumulation cannot account for the 6

receptor (BCR) -derived signals remains a matter of controversy http://www.jimmunol.org/ continued increase in pRb phosphorylation, which is maximal (4–6). Although B-1 cells resemble activated B-2 cells in terms of at 24 h. We show herein that PMA promotes the accumulation surface expression of IL-5R, CD44, and nuclear, activated STAT3 of functional cyclin D3-cdk4 complexes in B-1 cells following (7, 8), many additional molecular and transcriptional markers as- loss of cyclin D2. PMA also induces accumulation of cyclin sociated with B-2 cell activation are absent in B-1 cells (9, 10). D3-cdk4 complexes in B-2 cells; however, these complexes do B-1 cells differ significantly from B-2 cells in the signals re- not phosphorylate pRb. Thus, PMA is sufficient to induce syn- quired to induce proliferation. B-1 cells fail to enter in thesis and assembly of cyclin D3-cdk4 complexes in B-1 and response to anti-Ig, whereas B-2 cells are mitogenically stimulated B-2 cells; however, PMA triggers cyclin D3-cdk4 activation by BCR cross-linking (11, 12). Treatment with phorbol ester alone only in B-1 cells. These results reveal a novel regulatory step is sufficient to stimulate B-1 cells to enter S phase, whereas B-2 by guest on October 2, 2021 that controls activation of cyclin D3-cdk4 complexes whose cells exit quiescence, but subsequently arrest in of the function segregates differentially in B cell subsets. The Jour- (progression to S phase requires a second signal pro- vided by calcium ionophore) (11, 12). The molecular basis under- nal of Immunology, 2001, 166: 4273–4277. lying the unique proliferative response of B-1 cells to phorbol esters is not completely understood. -1 cells constitute a unique subset of B lymphocytes, dis- It is generally considered that growth signals regulate mamma- tinguished from conventional B lymphocytes (B-2) by lian cell cycle entry by stimulating the accumulation of D-type B numerous phenotypic and functional characteristics (re- (cyclins D1, D2, and D3) that function to activate a subset viewed in Refs. 1–3). As an example, B-1 cells localize primarily of cyclin-dependent kinases (cdks) (reviewed in Ref. 13). The ret- inoblastoma gene product (pRb) is a target of cdks and acts to

suppress G1-to-S phase progression (14–16). pRb is presently the *Department of Biology, Boston College, Chestnut Hill, MA 02467; and Departments most plausible candidate for regulating progression through the † ‡ § of Medicine and Microbiology, and The Evans Memorial Department of Clinical restriction (R) point (14, 16). A current model holds that pRb sup- Research, Boston University Medical Center, Boston, MA 02118 pression is alleviated through hyperphosphorylation that is medi- Received for publication December 8, 2000. Accepted for publication January 31, 2001. ated by both and D-type cyclin kinase complexes (15, 16). The costs of publication of this article were defrayed in part by the payment of page The proper timing and extent of cdk activation is controlled by charges. This article must therefore be hereby marked advertisement in accordance dephosphorylation of inhibitory sites, phosphorylation of activat- with 18 U.S.C. Section 1734 solely to indicate this fact. ing sites, the action of two families of cdk inhibitors (Ink4 and 1 This work was supported by Grant MCB-9603784 awarded by the National Science Cip/Kip family), and by cyclin binding (17). For example, D-type Foundation (to T.C.C.) and U.S. Public Health Service Grant AI29690 awarded by the National Institutes of Health (to T.L.R.). cyclins and cyclin E function as positive regulatory subunits for cdk4/6 and cdk2, respectively (13). The requirement of mamma- 2 Current address: Genaissance Pharmaceuticals, Five Science Park, New Haven, CT 06511. lian cyclins D1 and D2 in G1 phase progression has been defini- 3 Current address: Genetics Computer Group, 575 Science Drive, Madison, WI tively established (18, 19). In keeping with this, distinct pheno- Ϫ Ϫ Ϫ Ϫ 53719. types have been reported in / and cyclin D2 / mice 4 Current address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109. 5 Address correspondence and reprint requests to Dr. Thomas C. Chiles, Department of Biology, Boston College, 414 Higgins Hall, Chestnut Hill, MA 02467. E-mail 6 Abbreviations used in this paper: BCR, B cell Ag receptor; pRb, retinoblastoma address: [email protected] gene product; cdk, cyclin-dependent kinase; R point, .

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

● 4274 CUTTING EDGE

(20, 21). Recent studies suggest that cyclin D3 may function to material was removed by centrifugation, and the supernatant was incubated limit the rate of G phase progression (18, 22). Interestingly, in with 1.5 ␮g nonimmune rabbit IgG, 1.5 ␮g of anti-cdk4, or 1.5 ␮gof 1 ␮ cyclin D2Ϫ/Ϫ mice, B-2 cells appear to remain responsive to mi- anti-cdk6 Abs. After 3 h, 50 l of a 1:1 slurry of G-agarose was added and incubated for 1 h. The immune complexes were recovered by togenic signals due to a compensatory induction of cyclin D3 lev- centrifugation and washed six times with Rb buffer and then three times in els (23). a buffer of 50 mM HEPES, pH 7.4, and 1 mM DTT. The immune Cell cycle progression to S phase in normal B-2 cells requires complexes were resuspended in 30 ␮l of Rb kinase buffer (50 mM HEPES, the accumulation of cyclin D2 and cdk4 and to a lesser extent cdk6 pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 2.5 mM EGTA, 10 mM ␤ ␮ ␥ 32 -glycerophosphate, 0.1 mM Na3VO4, and 10 Ci [ P]ATP at 6000 (24–26). B-2 cells from xid mice, which exhibit aborted activation Ci/mmol) in the presence of 1 ␮g of a truncated Rb protein substrate in response to BCR cross-linking, do not up-regulate cyclin D2 (p56Rb). After 15 min at 30°C, the reactions were terminated by the protein, suggesting that accumulation of cyclin D2 in B-2 cells addition of 2ϫ SDS sample buffer, and the reaction products were sepa- may be linked to passage through the R point (26). Interestingly, rated through a 10% polyacrylamide SDS gel. Phosphorylated Rb was Solvason et al. (27) recently demonstrated a requirement for cyclin detected by autoradiography of the dried gel. D2 expression in CD5 B cell development. We have previously Immunoblot analysis demonstrated that phorbol ester induces unusually early and tran- sient expression of cyclin D2 in B-1, but not in B-2 cells (28). As For the detection of cyclins D2 and D3, B lymphocytes were solubilized in 100 ␮l Triton X-100 buffer (50 mM HEPES, pH 7.4, 15 mM EGTA, 137 such, we proposed that the early induction of cyclin D2 may ac- ␤ mM NaCl, 15 mM MgCl2, 0.1% Triton X-100, 10 mM -glycerophos- ␮ count for the rapid entry of B-1 cells into the cell cycle following phate, 1 mM Na3VO4, 1 mM PMSF, and 1 g/ml aprotinin/leupeptin); for phorbol ester stimulation. However, the transient nature of cyclin detection of endogenous pRb, B cells were solubilized in 100 ␮l Nonidet D2 expression in phorbol ester-stimulated B-1 cells suggests that it P-40 buffer containing 20 mM NaF (28). Insoluble debris was removed by

ϫ ␮ Downloaded from is unlikely to be responsible for progression through the G centrifugation at 15,000 g (15 min), and 10–20 g of total protein was 1/S tran- separated by polyacrylamide SDS gel electrophoresis and transferred to sition. Herein, we report experiments demonstrating that in phor- Immobilon-P membrane. Immune detection was conducted as previously bol ester-stimulated B-1 cells, cyclin D3-cdk4 complexes assemble described (24).

and are active pRb protein kinases in late G1 phase of the cell cycle. By contrast, phorbol ester stimulation of splenic B-2 cells Reagents Ј results in the assembly of cyclin D3-cdk4 complexes; however, F(ab )2 of goat anti-mouse IgM was obtained from Jackson ImmunoRe- these complexes fail to phosphorylate pRb in vitro. These findings search (West Grove, PA). PMA and the calcium ionophore, ionomycin, http://www.jimmunol.org/ constitute the first demonstration in a primary mammalian cell that were obtained from Sigma. Human pRb mAb (clone G3-245) was obtained from PharMingen (San Diego, CA). Anti-rabbit and anti-mouse IgG-con- -cdk complex assembly and activation can be dissociated jugated HRP Abs and anti-cdk4 Ab (sc-260) were obtained from Santa and regulated by different signals. Cruz Biotechnology (Santa Cruz, CA). Anti-cdk6 Ab (13446E) was ob- tained from PharMingen. Protein G-agarose was obtained from Life Tech- nologies (Gaithersburg, MD). Mouse anti-cyclin D2 Ab (DCS-3 and Materials and Methods DCS-5) and anti-cyclin D3 Ab (DCS-22) were a gift from Jiri Bartek (Di- Animals vision of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark) (19). The truncated Rb substrate protein (p56Rb) was obtained from QED Male BALB/cByJ mice at 8–14 wk of age were obtained from The Jackson Advanced Research Technologies (San Diego, CA). Enhanced chemilumi- by guest on October 2, 2021 Laboratory (Bar Harbor, ME). Mice were cared for and handled at all times nescence reagents were obtained from Kirkegaard & Perry Laboratories in accordance with National Institutes of Health and institutional (Gaithersburg, MD). guidelines.

B cell purification Results and Discussion We previously demonstrated that B-1 cell stimulation with the B-1 and B-2 lymphocytes were prepared by negative selection from peri- phorbol ester, PMA, produced cyclin D2 accumulation in a rapid toneal washout cells and from spleen cell suspensions, respectively, as described (9). The recovered B-1 cell population was 90–96% sIgMϩ, and transient manner (28). The initial onset of Cdk4-mediated pRb 780 CD5/Mac-1ϩ by flow cytometric analysis. The B cells were cultured at phosphorylation on Ser correlated with the accumulation and

37°C with 5% CO2 in RPMI 1640 medium (BioWhittaker, Walkersville, assembly of cyclin D2 into higher order complexes containing MD) supplemented with 10% heat-inactivated FBS (Sigma, St. Louis, cdk4. However, we noted that phosphorylation of pRb continued ␮ MO), 10 mM HEPES (pH 7.2), 50 M 2-ME, 2 mM L-glutamine, 100 to increase as B-1 cells progressed through the G -to-S phase tran- U/ml penicillin, and 100 ␮g/ml streptomycin. 1 sition. Interestingly, the maximal level of pRb phosphorylation

Immunoprecipitation occurred near the G1/S transition at a time when cyclin D2 was not expressed. These findings suggested that PMA stimulation of B-1 B cells were solubilized in 1 ml Nonidet P-40 buffer (50 mM Tris-HCl, pH cells promotes the accumulation of additional G -cyclin com- 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 2.5 1 ␮ ␤ plexes capable of phosphorylating pRb. To investigate the nature g/ml leupeptin/aprotinin, 1 mM Na3VO4, and 10 mM -glycerophos- phate) for 30 min (4°C) (28). Insoluble debris was removed by centrifu- of G1-cyclin complexes that might contribute to the phosphoryla- gation at 15,000 ϫ g for 15 min (4°C). The detergent-soluble cell lysates tion of endogenous pRb in late G phase, we evaluated the ex- ␮ ␮ 1 were incubated for 3 h with 1.5 g nonimmune IgG or 1.5 g anti-cdk4 pression of cyclin D3 in B-1 cells, noting that cyclin D1 is not Ab, or 1.5 ␮g anti-cdk6 Ab, followed by the addition of 50 ␮lofa1:1 slurry of protein G-agarose. After 90 min, the immune complexes were expressed in murine B lymphocytes (24–26). B-1 cells were cul- collected, washed several times in Nonidet P-40 buffer, and separated by tured in medium alone or stimulated with PMA for various times; electrophoresis through a 10% polyacrylamide SDS gel. The were cells were then collected and detergent-solubilized proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and im- separated by SDS-PAGE and immunoblotted with an anti-cyclin munoblotted with an anti-cyclin D3 mAb (1:500 dilution in TBST) as D3 mAb (Fig. 1). Cyclin D3 was not detected in B cells cultured described below. in medium alone. Stimulation of B-1 cells with PMA induced the Immune complex kinase assays accumulation of cyclin D3 at the 17- and 24-h time points. No cyclin D3 was expressed after PMA stimulation for 4 h, the point B cells were sonicated (4°C) in 1 ml Rb buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween at which cyclin D2 expression peaked (28), indicating that PMA- 20, 10% glycerol, 0.1 mM PMSF, 1 ␮g/ml leupeptin/aprotinin, 10 mM stimulated accumulation of cyclin D3 and cyclin D2 are distinct. ␤ -glycerophosphate, 1 mM NaF, and 0.1 mM Na3VO4) (29). Insoluble The accumulation of cyclin D3 paralleled peak endogenous pRb The Journal of Immunology 4275

FIGURE 1. Cyclin D3 expression is induced late during G1 phase in B-1 lymphocytes treated with PMA. Primary B-1 and B-2 lymphocytes were cultured in the presence of medium alone (M) or were stimulated with FIGURE 3. PMA promotes the assembly of cyclin D3-cdk4 complexes PMA (300 ng/ml) for 4, 17, and 24 h. At the indicated times, whole cell in both B-1 and B-2 lymphocytes. B-1 and B-2 cells were cultured in lysates were prepared in a 0.1% Trition X-100 buffer, and 10 ␮g protein medium alone (M) or were stimulated with PMA at 300 ng/ml for 4 and was resolved by 10% SDS-PAGE, transferred to Immobilon-P membrane, 24 h as indicated. Nonidet P-40 detergent extracts were prepared and se- and immunoblotted with a cyclin D3-specific mAb. The position of cyclin quentially immunoprecipitated (IP) with 1.5 ␮g of nonimmune serum (data D3 is indicated by the arrow. not shown), 1.5 ␮g of anti-cdk4 Ab, and 1.5 ␮g of anti-cdk6 Ab. The resulting immune complexes were resolved by 10% SDS-PAGE, trans- ferred to Immobilon-P membrane, and immunoblotted (IB) with anti-cyclin phosphorylation in PMA-stimulated B-1 cells (Fig. 2). PMA also D3 mAb. The position of cyclin D3 is indicated by the arrows. induced the accumulation of cyclin D3 in B-2 cells; however, in

contrast to B-1 cells, PMA stimulation of B-2 cells at parallel time Downloaded from points (i.e., 4, 16, 24 h) did not lead to increased pRb phosphor- were stimulated with PMA for 4 and 24 h, at which times cells ylation. As a positive control for these experiments, pRb phos- were detergent-solubilized and immunoprecipitated with rabbit phorylation was induced in B-2 cells stimulated with several dif- anti-mouse cdk4 or anti-mouse cdk6 Abs (Fig. 3). The immune ferent mitogens, including 25 ␮g/ml LPS, 10 ␮g/ml anti-Ig and the complexes were separated by SDS-PAGE and immunoblotted combination of PMA (300 ng/ml) plus ionomycin (400 ng/ml) for with anti-cyclin D3 mAb. PMA stimulation of B-1 cells induced 24 and 36 h (Fig. 2). the assembly of cyclin D3-cdk4/6 complexes, as evidenced by http://www.jimmunol.org/ The timing of cyclin D3 accumulation in B-1 cells suggests that the appearance of cyclin D3 in cdk4 and cdk6 immunoprecipi- it may contribute to the phosphorylation of endogenous pRb. To tates at 24 h. Surprisingly, the same was true of PMA-stimu- test this further, we sought to determine whether PMA promotes lated B-2 cells. No detectable cyclin D3 was present in cdk4/ the assembly and activation of cyclin D3-cdk complexes at a time 6-immune complexes isolated from unstimulated B-1 or B-2 commensurate with peak PMA-induced phosphorylation of endog- cells, and in parallel experiments, cyclin D3 was not detected in enous pRb. B-1 and B-2 cells were cultured in medium alone or any sample after immunoprecipitation with nonimmune serum (data not shown). Importantly, cyclin D3-cdk4/6 holoenzyme complexes were not detected in B-1 cells stimulated with PMA

for 4 h, which is consistent with the lack of detectable cyclin D3 by guest on October 2, 2021 expression at this time (see Fig. 1). To determine whether the PMA-induced cdk4- and cdk6-con- taining complexes were functional, Rb protein phosphorylation ac- tivity in cdk4 and cdk6 immunoprecipitates was analyzed using a recombinant COOH-terminal-truncated Rb protein as substrate (29). In extracts prepared from B-1 cells stimulated with PMA for 24 h, Rb phosphorylation was produced by cdk4 immune com- plexes and to a much lesser extent by cdk6 immune complexes (Fig. 4). Nonimmune complexes were devoid of Rb kinase activity

FIGURE 2. PMA treatment of B-1 cells, but not B-2 cells, promotes endogenous pRb phosphorylation. B-1 lymphocytes were cultured in me- dium alone (M) or were stimulated with medium containing PMA at 300 FIGURE 4. Cdk4- and cdk6-containing complexes produced by PMA ng/ml for 4, 16, and 24 h as indicated. As a positive control for pRb treatment are active Rb kinases in B-1, but not B-2 lymphocytes. B-1 and phosphorylation, B-1 cells were also stimulated with 25 ␮g/ml LPS for B-2 cells were cultured in medium alone (Med) or were stimulated with 24 h. B-2 cells were cultured in medium alone (M) or were stimulated with medium containing PMA at 300 ng/ml for the indicated times. Detergent medium containing 25 ␮g/ml LPS for 24 h or 10 ␮g/ml anti-Ig (aIg) or 300 lysates were prepared and sequentially immunoprecipitated (IP) with non- ng/ml PMA plus 400 ng/ml ionomycin (P/I) for 24 and 36 h as indicated. immune serum (data not shown), anti-cdk4 Ab, and anti-cdk6 Ab, and the Nonidet P-40 detergent lysates were prepared, and 10 ␮g protein was re- immune complexes were recovered and assayed for in vitro kinase activity solved by 7.5% SDS-PAGE, transferred to Immobilon-P membrane, and using Rb-GST fusion protein (p56Rb) substrate as described in Materials immunoblotted with an anti-human pRb Ab. The position of pRb is indi- and Methods. B-2 cells were also stimulated with 300 ng/ml PMA plus 400 cated by the arrow. The molecular weight of standard proteins is given in ng/ml ionomycin (P/I) for 24 h. The position of the phosphorylated Rb- kilodaltons and indicated on the left. GST fusion protein is indicated by the arrows. 4276 CUTTING EDGE

(data not shown). In parallel experiments, Rb phosphorylation At present the nature of the regulatory step necessary to promote was not stimulated above control by cdk4 or cdk6 immune com- activation of assembled cyclin D3-cdk complexes in B-2 cells is plexes isolated from PMA-stimulated B-2 cells. As a positive unknown. Given the presence of assembled D-type cyclin-cdk control, cdk4 and to a lesser extent cdk6 immune complexes complexes, it is unlikely that formation of inactive binary cdk/Ink4 recovered from B-2 cells stimulated with the combination of complexes or decreased expression of D-type cyclin or cdk ac- PMA plus calcium ionophore exhibited inducible Rb kinase ac- counts for the observed inhibition of cyclin D3-cdk complex ac- tivity (Fig. 4, lane P/I). tivity in B-2 cells (13, 17). PMA treatment of B-2 cells does not We previously reported that cyclin D2 accumulates rapidly and alter the relative amount of D-type cyclin-associated p21Cip1 and in a transient manner following stimulation of B-1 cells with PMA p27Kip1 proteins in comparison to control cells (data not shown). alone (28). Commensurate with cyclin D2 accumulation, B-1 cells Thus, regulation of cyclin D3-cdk complex activity might depend express phosphorylated pRb on Ser780, which increases in a time- on a previously uncharacterized enhancing protein, a previously dependent manner. These results suggest that cyclin D2 plays an uncharacterized inhibitory protein, or a posttranslational modifi- cation of the D-type cyclin and/or cdk4/6 subunits (e.g., phosphor- important role in early G1 phase progression in B-1 cells during PMA-mediated proliferation. Although our previous findings in ylation on an as yet unmapped amino acid residue) induced by B-1 cells are consistent with the emerging view that cyclin D2 is PMA in one B cell population and not the other. Studies are pres- ently underway to further understand the molecular step(s) that the primary G1 cyclin involved in passage through the R point during BCR-mediated B-2 cell proliferation (23–26), we note that regulate cyclin D3-cdk complex activation in B lymphocytes. the majority of pRb phosphorylation occurs in mid-to-late G1

phase and at a time during which cyclin D2 is not detected in B-1 Downloaded from Acknowledgments cells by Western blot analysis (28). This observation points to the We thank Dr. Jiri Bartek (Division of Cancer Biology, Danish Cancer presence of additional G cyclins that contribute to pRb phosphor- 1 Society, Copenhagen, Denmark) for the murine D-type cyclin mAbs. ylation following loss of detectable cyclin D2. The results pre- sented herein indicate that cyclin D3 accumulates in response to PMA stimulation of B-1 cells. Furthermore, the timing of cyclin References D3 accumulation, which was distinct from that of cyclin D2, sug- 1. Herzenberg, L. A. 2000. B-1 cells: the lineage question revisited. Immunol. Rev. http://www.jimmunol.org/ gests the involvement of cyclin D3 in PMA-induced mid-to-late 175:9. 2. Morris, D. L., and T. L. Rothstein. 1994. CD5ϩ B (B-1) cells and immunity. In G1 phase progression. Consistent with this conclusion, cyclin D3 Handbook of B and T Lymphocytes, E. C. Snow, ed. Academic, San Diego, CA, assembled into higher order complexes containing both cdk4 and p. 421. 3. Hayakawa, K., and R. R. Hardy. 2000. Development and function of B-1 cells. cdk6 in response to PMA in B-1 cells. Furthermore, cdk4 and to a Curr. Opin. Immunol. 12:346. lesser extent cdk6 immune complexes from PMA-stimulated B-1 4. Stall, A. M., M. C. Farinas, D. M. Tarlington, P. A. Lalor, L. A. Herzenberg, cells were capable of phosphorylating Rb in vitro at levels that S. Strober, and L. A. Herzenberg. 1988. Ly-1 B-cell clones similar to human chronic lymphocytic leukemias routinely develop in older normal mice and were substantially greater than parallel immune complexes recov- young autoimmune (New Zealand Black-related) animals. Proc. Natl. Acad. Sci. ered from control cells. Taken together, these results suggest a role USA 85:7312. Ϫ for cyclin D3-cdk4 holoenzyme in passage through the G /S phase 5. Cong, Y. Z., E. Rabin, and H. H. Wortis. 1991. Treatment of murine CD5 B by guest on October 2, 2021 1 cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation path- transition during PMA-mediated B-1 cell proliferation. In keeping ways. Int. Immunol. 3:467. with our findings herein, mounting evidence in several cell types 6. Arnold, L. W., C. A. Pennell, S. K. McCray, and S. H. Clarke. 1994. Develop- ment of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 supports a role for cyclin D3 function in the control of mammalian population occurs after immunoglobulin . J. Exp. Med. 179:1585. cell G1/S transition (19, 22, 30). 7. Hitoshi, Y., N. Yamaguchi, S. Mita, E. Sonoda, S. Takaki, A. Tominaga, and K. Takatsu. 1990. Distribution of IL-5 receptor-positive B cells: expression of The accumulation and assembly of substantial levels of cyclin ϩ IL-5 receptor on Ly-1(CD5) B cells. J. Immunol. 144:4218. D3-containing cdk complexes in B-2 cells following PMA stimu- 8. Karras, J. G., Z. Wang, L. Huo, R. G. Howard, D. A. Frank, and T. L. Rothstein. lation was unexpected. The absence of pRb kinase activity asso- 1997. STAT3 is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes. J. Exp. Med. 185:1035. ciated with these complexes in B-2 cells suggests that an additional 9. Morris, D. L., and T. L. Rothstein. 1993. Abnormal transcription factor induction signal(s) is (are) required for function and points to the existence through the surface immunoglobulin M receptor of B-1 lymphocytes. J. Exp. of a hitherto unknown regulatory step in B cells that controls the Med. 177:857. 10. Wang, Z., D. L. Morris, and T. L. Rothstein. 1995. Constitutive and inducible activation of preformed cyclin D3-cdk complexes. It is well es- levels of egr-1 and c-myc early growth response gene expression in B-1 lym- tablished that formation of cyclin D holoenzyme complexes relies phocytes. Cell. Immunol. 162:309. 11. Rothstein, T. L., and D. L. Kolber. 1988. Peritoneal B cells respond to phorbol upon growth factor signals that act both transcriptionally, to induce esters in the absence of co-mitogen. J. Immunol. 140:2880. accumulation of D-type cyclin and cdk, and posttranslationally, to 12. Rothstein, T. L., and D. L. Kolber. 1988. Anti-Ig antibody inhibits the phorbol promote cyclin D-cdk assembly (17, 30). For example, ectopically ester-induced stimulation of peritoneal B cells. J. Immunol. 141:4089. 13. Sherr, C. J., and J. M. Roberts. 1995. Inhibitors of mammalian G1 cyclin-depen- expressed cyclin D1 and cdk4 subunits are not active in NIH 3T3 dent kinases. Dev. 9:1149. cells in the absence of serum because they fail to assemble in the 14. Weinberg, R. A. 1995. The retinoblastoma protein and cell cycle control. Cell 81:323. absence of mitogenic signals. The mitogenic signal for assembly 15. Hatakeyama, M., J. A. Brill, G. R. Fink, and R. A. Weinberg. 1994. Collaboration can be provided by activation of the Ras/Raf-1/Erk pathway or by of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes ectopic expression of MEK1 (30). Thus, growth factor signals not Dev. 8:1759. 16. Lundberg, A. S., and R. A. Weinberg. 1998. Functional inactivation of the reti- only function to induce cyclin D1 transcription, but also to pro- noblastoma protein requires sequential modification by at least two distinct cy- mote assembly of cyclin D1 into cdk4-containing catalytically ac- clin-Cdk complexes. Mol. Cell. Biol. 18:753. tive complexes. Our findings in B-2 cells suggest that accumula- 17. Sherr, C. J., and J. M. Roberts. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 15:1501. tion and assembly of cyclin D3-dependent kinases is not sufficient 18. Quelle, D. E., R. A. Ashmun, S. A. Shurtleff, J.-Y. Kato, D. Bar-Sagh, to induce kinase activation. To our knowledge, this constitutes the M. F. Roussel, and C. J. Sherr. 1993. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 7:1559. first demonstration in a primary (not genetically engineered) mam- 19. Bartkova, J., J. Lukus, M. Strauss, and J. Bartek. 1998. Cyclin D3: requirement malian cell that cyclin D-cdk accumulation/assembly and D-type for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation. Oncogene 17:1027. cyclin-cdk activation can be dissociated and are regulated by dif- 20. Sicinski, P., J. L. Donaher, Y. Geng, S. B. Parker, H. Gardner, M. Y. Park, ferent signals. R. L. Robker, J. S. Richards, L. K. McGinnis, J. D. Biggers, et al. 1996. Cyclin The Journal of Immunology 4277

D2 is an FSH-responsive gene involved in gonadal cell proliferation and onco- 26. Brorson, K., M. Brunswick, S. Ezhevesky, D. G. Wei, R. Berg, D. Scott, and genesis. Nature 384:470. K. E. Stein. 1997. xid affects events leading to B cell cycle entry. J. Immunol. 21. Sicinski, P., J. L. Donaher, S. B. Parker, T. Li, A. Fazeli, H. Gardner, 159:135. S. Z. Haslam, R. T. Bronson, S. J. Elledge, and R. A. Weinberg. 1995. Cyclin D1 27. Solvason, N., W. W. Wu, D. Parry, D. Mahony, E. W.-F. Lam, J. Glassford, provides a link between development and oncogenesis in the retina and breast. G. G. B. Klaus, P. Sicinski, R. Weinberg, Y. J. Liu, et al. 2000. Cyclin D2 is Cell 82:621. essential for BCR-mediated proliferation and CD5 B cell development. Int. Im- 22. Herzinger, T., and S. I. Reed. 1998. Cyclin D3 is rate limiting for the G1/S phase munol. 12:631. transition in fibroblasts. J. Biol. Chem. 273:14958. 28. Tanguay, D. A., T. P. Colarusso, S. Pavlovic, M. Irigoyen, R. G. Howard, 23. Lam, E. W.-F., J. Glassford, L. Banerji, N. Shaun, B. Thomas, P. Sicinski, and J. Bartek, T. C. Chiles, and T. L. Rothstein.; 1999. Early induction of cyclin D2 G. G. B. Klaus. 2000. Cyclin D3 compensates for loss of cyclin D2 in mouse B- expression in phorbol ester-responsive B-1 lymphocytes. J. Exp. Med. 189:1685. lymphocytes activated via the antigen receptor and CD40. J. Biol. Chem. 275:3479. 24. Tanguay, D. A., and T. C. Chiles. 1996. Regulation of the catalytic subunit 29. Matsushime, H., D. E. Quelle, S. A. Shurtleff, M. Shibuya, C. J. Sherr, and J.-Y. (p34PSK-J3/Cdk4) for the major D-type cyclin in mature B cells. J. Immunol. Kato. 1994. D-type cyclin dependent kinase activity in mammalian cells. Mol. 156:539. Cell. Biol. 14:2066. 25. Solvason, N., W. W. Wu, N. Kabra, X. Wu, E. Lees, and M. C. Howard. 1996. 30. Cheng, M., V. Sexl, C. J. Sherr, and M. F. Roussel. 1998. Assembly of cyclin Induction of cell cycle regulatory proteins in anti-Ig stimulated mature B lym- D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated pro- phocytes. J. Exp. Med. 184:407. tein kinase kinase (MEK1). Proc. Natl. Acad. Sci. USA 95:1091. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021