The p h o sp h o in o sitid e s are a sm all g roup o f m e m b ran e p h o s p h o lip id s w h ic h are u n iq u e in th at th e ir m y o - in o s ito l
C hapter 1________________________ Introduction________________________________3 2 headgroup can be phosp h o ry lated at m ultiple sites (122). They form a m in o r c o m p o n e n t o f m o st, i f not all, e u k a ry o tic m em b ran es w ith the three m ost p red o m in an t p h o sp h o in o sitid e s b ein g p h o sp h a tid y lin o sito l [Ptdlns] w hich form s the m ajo rity , p h o s p h a t i d y l i n o s i t o l 4 - m o n o p h o s p h a t e [ P t d I n s ( 4 ) P ] a n d p h o sp h a tid y lin o sito l 4 ,5 -b isp h o sp h a te [P tdIns(4,5)P 2].
There is considerable evidence that stim ulation o f inositol p h o s p h o lip id m e ta b o lism v ia a c tiv a tio n o f p h o s p h o in o s itid e - specific phospholipase C (PI-PLC) is a m ajor signal transduction p a th w ay for m any e x tra ce llu lar sig n allin g m o lecu les in clu d in g h o rm o n e s, p o ly p e p tid e g ro w th fac to rs, n e u r o tr a n s m itte r s and im m u n o g lo b u lin s (123). P L C -c a ta ly z e d I n s ( l ,4 ,5 ) P 3 fo rm a tio n m obilizes Ca^+ by binding to specific intracellular receptors. These receptors prom ote the opening o f calcium channels in v esicu lar storage sites associated with the endoplasmic reticulum (123).
The In s(l,4 ,5 )P 3 signal produced by receptor m ediated PLC a c tiv a tio n is sw itc h e d o f f by se q u e n tia l p h o s p h o r y la tio n o f In s(l,4 ,5 )P 3 on the 3-position, follow ed by dep h o sp h o ry latio n at the 5-position. Thus, Ins(l,4,5)P3 is m etabolized by two enzymes, a 5-phosphatase and a 3-kinase. The first converts In s(l,4 ,5 )P 3 to I n s ( l,4 ) P 2 and evidence indicates that the m etabolite I n s ( l,4 ) P 2 do not mobilize Ca2+. In s(l,4 )P 2 is further dephosphorylated to Ins by phosphatases that are inhibited by Li+. The second enzym e converts In s(l,4 ,5 )P 3 to In s(l,3 ,4 ,5 )P 4 which some reports suggest is also in v o lv ed in Ca^+ fluxes. I n s ( l ,3 ,4 ,5 ) P 4 can also be d e p h o s p h o r y la te d by 3 -p h o s p h a ta s e s and 5 - p h o s p h a ta s e s and eventually back to Ins, which can be used for the regeneration o f inositol lipids (reviewed in 124, 125).
Interestingly, PtdIns(4,5)P2 as well as being a substrate for PLC, is thought to play a role in actin cytoskeleton regulation (see section 1.9.1.2 o f Introduction). PtdIns(4,5)P2 was identified as a functional ligand for PH dom ains and therefore it serves as a m em brane attachm ent site for proteins with this domain. O f note, th e h y d r o ly s is o f p h o s p h a tid y lin o s ito l (P I) b y th e h u m a n p h o sp h o lip ase C51 was found to be regulated by the PH domain o f th is en zy m e. F u rth e rm o re the a c tiv ity o f this e n z y m e was s tim u la te d by P td In s(4 ,5 )P 2 in a d o se -d e p e n d e n t m a n n e r and stim u latio n was inhibited by In s (l,4 ,5 )P 3 in the same m anner. These results suggest a general m echanism for the regulation o f
C hapter 1________________________ Introduction________________________________3 3 other p ro tein s by PtdIns(4,5)P2 (126). T herefore, P td In s(4 ,5 )P 2 depletion by PLC -m ediated hydrolysis could also have im portant c o n seq u e n ce s for the cell in clu d in g in itia tio n o f c y to s k e le ta l rearrangem ent (see section 4.11 o f Results and Discussion).
1 . 6 . 1 CALCIUM
The levels o f Ca^+ inside most cells is typically about 10-^M, w here its c o n ce n tra tio n in the e x tra c e llu la r fluids is u su a lly g rea ter than lO-^M. E ukaryotic cells also have a sp e c ia liz e d in tr a c e llu la r c a lc iu m - s e q u e s te r in g c o m p a rtm e n t in w h ic h the level o f Ca2+ is very high. Thus, there is a large gradient tending to drive Ca^+ into the cytosol across the plasm a m em brane and across the m em brane o f the intracellular compartm ents.
Increase in the intracellular concentration o f Ca^+ caused by g ro w th fa c to rs, h o rm o n e s or e le c tric a l sig n a ls can tr ig g e r responses as varied as contraction, secretion, cellular proliferation and changes in gene expression. This effect is extremely rapid and may be observed as little as 15 seconds after addition o f growth factors. Purified grow th factors including PDGF, b o m b esin and vasopressin have also been found to stimulate efflux o f Ca^+ from an intracellular store(s) in Swiss 3T3 cells (127-130).
G enerally, recep to r activated Ca^+ m obilization occurs via the inositol phosphate-Ca2+ signalling system. It involves a Ca^ + transient increase from a limited intracellular ([Ca^+Ji) pool and a more prolonged phase o f extracellular Ca^+ entry. Subsequent to the tran sien t increase in cytosolic Ca^+ co ncentration a C a2 + - A T Pase rem oves Ca^+ from the cell (21). The available evidence in d icates th at the initial internal Ca^+ release is sig n aled by I n s ( l ,4 ,5 ) P 3 . The m ec h an ism by w h ic h I n s ( l ,4 ,5 ) P 3 re le a s e s i n t r a c e l l u l a r Ca^+ in v o lv es its in te ra c tio n w ith a sp e cific intracellular m embrane receptor and opening o f a Ca^+ channel on the responsive organelle.
Changes in intracellular Ca^+ is an important signal for the reg u latio n o f cytoskeletal reorganization. Ca^+ achieves this and other effects by binding to proteins and changing their activity. It has a direct effect on the plasma m em brane through its influence on io n c h a n n e ls an d m e m b ra n e b o u n d e n z y m e s su c h as p h o s p h o lip a s e s , b o th o f w h ich th en h ave an in f lu e n c e on c y to sk e le ta l structures. In the cytosol Ca^+ in teracts w ith the
C hapter 1________________________ Introduction________________________________3 4 proteolytic enzyme calpain, with protein kinase C (see below ) and calmodulin. Finally, Ca^+ can bind directly to cytoskeletal proteins such as a -a c tin in and gelsolin and change their activity.
S i g n i f i c a n t l y , Ca^+ has b een im p lica te d as a se co n d "co stim u lato ry " event in ty ro sin e p h o sp h o ry la tio n o f F A K in platelets stimulated with epinephrine (see below and 131).