The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain (ϳ50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-⌬189, MPDE3B, and M3B-⌬196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-⌬321 and M3B-⌬302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-⌬397 and H3A-⌬457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-⌬510 and M3B-⌬604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-⌬196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-⌬607, H3A-⌬721, and M3B-⌬604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of ϳ50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminaltruncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-⌬189), was virtually identical to that of the wild type. M3B-⌬302 (lacking region 1) and H3A-⌬397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-⌬189. Proteins that lacked both regions 1 and 2, H3A-⌬510 and M3B-⌬604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.

No abstract

Mahmoud Darwish is considered the national Palestinian poet, a symbol of the national struggle against the Israeli occupation. Sami Shalom Chetrit and Almog Behar, two prominent Israeli poets of Arab decent (Chetrit was born in Morocco; Behar’s family is from Iraq), have both written poems directed to Darwish in which they address both his vast poetic corpus and his public and political figure. Close reading these poetic addresses, I discuss Darwish’s own poetry as an intertext in these Hebrew poems, as well as the significance of writing about him and to him in Hebrew and in Israel, specifically by poets of Arab descent. Moreover, this discussion serves as an opportunity to read Hebrew and Arabic together, challenging the clear-cut national distinctions while still acknowledging their pervasiveness and the inevitable questions of power, as the poems do themselves.