Abstract
The mammalian SWI/SNF chromatin remodeling complex is a heterogeneous collection of related protein complexes required for gene regulation and genome integrity. It contains a central ATPase (BRM or BRG1) and various combinations of 10-14 accessory subunits (BAFs for BRM/BRG1 Associated Factors). Two distinct complexes differing in size, BAF and the slightly larger polybromo-BAF (PBAF), share many of the same core subunits but are differentiated primarily by having either AT-rich interaction domain 1A/B (ARID1A/B in BAF) or ARID2 (in PBAF). Using density gradient centrifugation and immunoprecipitation, we have identified and characterized a third and smaller SWI/SNF subcomplex. We termed this complex GBAF because it incorporates two mutually exclusive paralogs, GLTSCR1 (glioma tumor suppressor candidate region gene 1) or GLTSCR1L (GLTSCR1-like), instead of an ARID protein. In addition to GLTSCR1 or GLTSCR1L, the GBAF complex contains BRD9 (bromodomain-containing 9) and the BAF subunits BAF155, BAF60, SS18, BAF53a, and BRG1/BRM. We observed that GBAF does not contain the core BAF subunits BAF45, BAF47, or BAF57. Even without these subunits, GBAF displayed in vitro ATPase activity and bulk chromatin affinity comparable to those of BAF. GBAF associated with BRD4, but, unlike BRD4, the GBAF component GLTSCR1 was not required for the viability of the LNCaP prostate cancer cell line. In contrast, GLTSCR1 or GLTSCR1L knockouts in the metastatic prostate cancer cell line PC3 resulted in a loss in proliferation and colony-forming ability. Taken together, our results provide evidence for a compositionally novel SWI/SNF subcomplex with cell type-specific functions.
Highlights
The Ca2؉/calmodulin-dependent protein kinase kinase  (CaMKK)/5-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca2؉-dependent metabolic pathways and cancer growth
Autonomous activity of recombinant CaMK kinase (CaMKK) is suppressed during AMPK activation
We first examined the activity of Escherichia coli-expressed CaMKK during a CaMKK-mediated AMPK activation reaction in which we incubated CaMKK with either wild-type or kinase-dead mutant (K45R) AMPK in the presence of Mg-ATP and EGTA for various time points (5– 60 min) followed by the withdrawal of constant amounts of reaction mixture into buffer containing excess EDTA to stop the phosphorylation reaction
Summary
CaMK, Ca2ϩ/calmodulin-dependent protein kinase; CaMKK, Ca2ϩ/CaM-dependent protein kinase kinase ; AID, autoinhibitory domain; CaM, calmodulin; AMPK, 5Ј-AMP–activated protein kinase; CDK5, cyclin-dependent kinase 5; GSK3, glycogen synthase kinase 3. Cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3) can phosphorylate multiple residues in the N-terminal regulatory domain (Ser129, Ser133, and Ser137 in human CaMKK), resulting in decreased autonomous activity [41]. This observation is in agreement with the finding that CaMKK/AMPK pathway activation requires Ca2ϩ/CaM signaling, whereas the CaMKK substrate AMPK is not Ca2ϩ/CaM-dependent [13,14,15, 32]. We identified a single AMPK phosphorylation site in the N-terminal regulatory domain of CaMKK that acts as a switch for Ca2ϩ/ CaM dependence and suggests a unique enzymatic mechanism of CaMKK/AMPK signaling cascade regulation
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