Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 3, 2000, pp. 192

Biotecnología Aplicada, Volume 17, July-September 2000, p. 192

Pleckstrin Homology Domains in Cell Signaling

Matti Saraste, Niklas Blomberg, Elena Baraldi, Michael Nilges

European MolecuIar Biology Laboratory, Meyerhofstrasse 1, Postfach 10.2209, D-39012 Heidelberg, Germany

From selection of papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: BA00056

Pleckstrin homology (PH) domains are a structurally conserved family that is associated with many regulatory pathways within the cell. In particular, PH domains are found in many proteins involved in signal transduction such as phospholipases, GTPase-regulating proteins and protein kinases, but also in cytoskeletal proteins such as spectrin and syntrophin. They generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. In some cases, PH domains can directly control enzymatic activity of adjacent kinase or nucleotide exchange domains [1].

Crystal structures of the PH domains from PLC-d1 and spectrin have been determined in complex with lns(1,4,5)P3 and that of the Btk PH domain in complex with Ins(1,3,4,5)P4 [see 1, 2]. The phospholipid-binding site is not structurally conserved in all cases. The spectrin domain binds phospholipid between b1/b2 and b5/b6 loops, whereas the ligand binds on the opposite side of the b1/b2 loop, between this and the b3/b4 loop in the other PH domains.

Many studies have shown that some PH domains are specific for 3-phosphorylated inositol derivatives and therefore represent possible downstream targets of phosphoinositide 3-kinase (PI 3-kinase). PI 3-kinase is activated by receptor tyrosine kinases and G-protein coupled receptors in response to a wide range of cellular stimuli and produces Ptdlns(3,4)P2 and Ptdlns (3,4,5)P3 on the inner leaflet of the plasma membrane. Several PH domains, such as those of Akt, Btk, PLCg, and ARNO, have been shown to translocate to the plasma membrane following PI 3-kinase activation.

Details of molecular mechanism of inositol binding can be gained from mutations in the Btk PH domain. These mutations lead to a defect in the maturation of B cells, resulting in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). The mutations may be grouped depending on their effect on binding. Many mutations directly perturb the inositol phosphate-binding site, whereas others have a more indirect effect. The PH domain mutants in which Arg28 is substituted with either a cysteine or a histidine remove a positive charge within the binding pocket and thus have a strongly reduced affinity for lns(1,3,4,5)P4.

Mutations located on the domain surface outside the binding pocket highlight the role of electrostatics in binding. Mutation of Lys 19, which is not in direct contact with the ligand, to a glutamate reverses a charge and significantly decreases the positive potential around the binding site. The resulting decreased affinity for the negatively charged inner surface of the cell membrane appears to be sufficient for the disease phenotype. Analogously, a gain of function mutant, E41K, enhances the positive potential. This hinders the removal of Btk from the membrane surface and hence its deactivation, leading to a transformation of cultured cells [2].

PH domains can regulate the activity of proteins not only by targeting to the correct subcellular location but recent data also suggest a direct allosteric activation. An example is the regulation of nucleotide exchange activity in the Dbl protein family by PH domains. Their structural hallmark is that the Dbl homology (DH) domain is immediately followed by a PH domain. The DH domain is a specific guanine nucleotide exchange factor (GEF) for the Rho family of small GTPases, which are involved in regulation of the actin cytoskeleton. Several studies show that the binding of inositol phosphates to the PH domain can modulate the nucleotide exchange activity of the adjacent DH domain. In addition, in many proteins the Sec7 domains that are GEFs acting on the Arf-class of small GTPases ad, have an adjacent PH domain. GRP1, ARNO, and cytohesin-1 that belong to this family specifically bind to 3-phosphorylated inositols and localize to the membrane following activation of PI 3-kinase [reviewed in 1].

A peculiar feature of the PH domain structures is the strong polarization of charges [3]. In the lipid-binding domains, the face of the molecule interacting with the inositol phosphate is surrounded by a strong positive potential. Electrostatic effects are probably involved in orientation of the molecule towards the membrane and can be a major determinant in binding. Electrostatic properties are in general well conserved within the PH domain family. However, in comparison to the domains that bind phospholipids, a small number of PH domains shows reversed polarization or an overall negative potential [1, 3]. That is inconsistent with binding to negatively charged phospholipids.

It is unclear whether all the DH-PH domains are regulated by phosphoinositides. Roughly half of the domains in the DH-PH family have the electrostatic properties typical for the PH domains with positively charged (lipid-) binding surface. For instance, the PH domain from Sos shows a similar potential profile to the main group of PH domains, and it is reported to bind phospholipids. In contrast, five out of seven PH domains within the whole family that are predicted to have a reversed potential with negative charge around the canonical lipid-binding site, are linked to a DH domain [3]. It is not likely that these domains bind acidic phospholipids.

References

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