Protein targeting to subnuclear higher order structures: A new level of regulation and coordination of nuclear processes

Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222– 230, 1998. © 1998 Wiley-Liss, Inc.     

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.     

ADAM12 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity

ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.     

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the -10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A37 for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A37-RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to -10 sequences, DNA sequence at -35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in -10 and -35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions.     

Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.