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Cell Signalling Biology Michael J. Berridge Module 2 Cell Signalling Pathways 2 43

• The Src homology (SH2) domains of the regulatory sub- early endosome protein sorting and intraluminal vesicle
unit enable the enzyme to associate with phosphotyr- formation (Module 4: Figure intraluminal endosomal ves-
osine residues on the cytoplasmic domains of recept- icle formation)and endosome vesicle fusion to early en-
ors such as the platelet-derived growth factor receptor dosomes (Module 4: Figure endosome vesicle fusion). In
(PDGFR) (Module 1: Figure PDGFR activation)orthe the case of autophagy, hVps34 plays a central role in driv-
CD19 B cell co-receptor (Module 9: Figure B cell activ- ing the early induction, nucleation and elongation steps
ation). responsible for forming the autophagic vacuole (Module
• The Src homology 3 (SH3) domain can bind to proline- 11: Figure autophagy signalling mechanisms).
rich regions of Shc, Cbl and dynamin.
• The proline-rich regions can bind to the SH3 domains
of Abl, Src, Lck, Lyn, Fyn and growth factor receptor PtdIns 4-kinase (PtdIns 4-K)
bound protein 2 (Grb2). PtdIns 4-kinase catalyses phosphorylation of PtdIns on the
• The p85β inhibitory subunit is a target for miR-126, 4-position of the inositol ring to produce PtdIns4P (Step
which plays an important role in the regulation of 2in Module 2: Figure phosphoinositide metabolism). En-
zyme activity is found in most cellular membranes (plasma
angiogenesis.
membrane, endoplasmic reticulum, Golgi, nuclear mem-
The Class IB enzyme has a different activation mechan- brane and secretory vesicles). Cells have two classes of
ism in that it is stimulated by heterotrimeric G proteins PtdIns 4-K: Type II and Type III.
(Module 2: Figure PtdIns 3-kinase signalling). It has a The Type II PtdIns 4-K, which contains wortmannin-
p110γ catalytic subunit and two regulatory subunits (p101 insensitive α-and β-isoforms, may be responsible for gen-
and p84) (Module 2: Figure PI 3-K family). These regulat- erating the lipid substrates in the membrane that are used
ory subunits may mediate the translocation of the p110γ for signalling. The enzymatic activity of PtdIns 4-kinase is
greatly enhanced in various cancer cells.
catalytic subunit to the membrane by binding to the G βγ
subunit. The Type III PtdIns 4-K, which has α-and β-isoforms,
The Class I enzymes interact strongly with the onco- is sensitive to the drug wortmannin. The PtdIns 4-KIIIα
gene Ras (Module 2: Figure PtdIns 3-kinase signalling)and found in the endoplasmic reticulum and the PtdIns 4-
this can have important consequences for both upstream KIIIβ found in the cytosol and Golgi are responsible
and downstream events, depending on the cellular context. for producing PtdIns4P, which is one of the localized
With regard to the former, the binding of Ras can enhance phosphoinositide lipid signalling molecules used for ves-
enzyme activation and the generation of lipid messengers. icle trafficking (Module 2: Figure localized inositol lipid
On the other hand, the association with Ras might mediate signalling). The PtdIns 4-KIIIβ at the Golgi is controlled
some of the downstream effects of the PtdIns 3-kinases. by the Arf signalling pathway (Module 2: Figure Arf sig-
Class I PtdIns 3-kinases are strongly inhibited by the nalling). It is the Type III PtdIns 4-K isoform that is ac-
fungal metabolite wortmannin (an irreversible inhibitor) tivated by the neuronal Ca 2 + sensor-1 protein (NCS-1)
and by LY294002 (a reversible inhibitor). to enhance exocytosis. Originally, it was considered that
PtdIns4P functioned solely as a precursor for the forma-
Class II PtdIns 3-kinase
tion of PtdIns4,5P 2 , but there is increasing evidence for a
The Class II PtdIns 3-kinase has a more restricted substrate role as part of a PtdIns4P signalling cassette.
range in that it phosphorylates just PtdIns and PtdIns4P.
There are no regulatory subunits, and the three catalytic
subunits (PI3KC2α, PI3KC2β and PI3KC2γ) are char- PtdIns phosphate kinases
acterized by having a C-terminal phox homology (PX) There is a family of PtdIns phosphate kinases that are di-
domain and a C2 domain (Module 2: Figure PI 3-K fam- vided into three subfamilies (Types I--III) that are localized
ily). to different compartments and function to phosphorylate
different phosphoinositides.
Class III PtdIns 3-kinase
The Class III PtdIns 3-kinase has a restricted substrate
range in that it phosphorylates just PtdIns to form PtdIns4P 5-kinase (PtdIns4P 5-K)
PtdIns3P. The prototype of this class is vacuolar pro- This versatile family of Type I PtdIns phosphate kinases
tein sorting 34 (Vps34) protein found in Saccharomyces (also abbreviated as PIPKI) is composed of three isoforms
cerevisiae of which there is a human homologue (hVps34). (α, β and γ) that are located in different cellular regions
The activity of hVps34 is regulated by Ca 2 + and this con- such as the plasma membrane, focal adhesions, Golgi and
tributes to the regulation of the target of rapamycin (TOR), nucleus. Their primary role is to phosphorylate PtdIns4P
which functions in cell growth control (see steps 4 and 5 in to PtdIns4,5P 2 (Step 8 in Module 2: Figure phosphoinos-
Module 9: Figure target of rapamycin signalling). There is itide metabolism).
aCa 2 + -dependent CaM-binding domain located between
residues 318 and 334 in the accessory domain (Module 2: PtdIns4P 5-kinase Iα
Figure PI3-K family). This enzyme functions at multiple locations in the cell. It is
The hVps34 plays an important role in intracellular traf- the major enzyme responsible for forming PtdIns4,5P 2 at
ficking such as the early endosome to plasma membrane the plasma membrane. It is also found within the nucleus
trafficking (Module 4: Figure early endosome budding), at nuclear speckles that are sites of mRNA processing.

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