Cutting Edge: Differential Signaling Requirements for Activation of Assembled Cyclin 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; ; doi: 10.4049/jimmunol.166.7.4273 Downloaded from http://www.jimmunol.org/content/166/7/4273 References This article cites 29 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/166/7/4273.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 D2-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 gene 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 S phase 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 G1 phase of the function segregates differentially in B cell subsets. The Jour- cell cycle (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 (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 cyclin E 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 cyclin D1 / 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, restriction 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 protein 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.