10 A niversary of IIMCB

a decrease in the complexity of dendritic arbors and
shrinkage of dendritic fields. Moreover, CLIP-170 knockdown
exerts a strong effect on the shape of dendritic arbor
even under conditions promoting dendritogenesis such
as the overexpression of constitutively active forms of PI3K
and Akt kinases, which are crucial upstream components of
mTOR signaling pathway. Taken together, this data strongly
suggests the role of CLIP170 in the development of dendritic
arbor, which may be regulated in mTOR dependent manner.
To support our hypothesis that mTOR is an important
regulator of microtubule dynamics and CLIP-170 serves as a
mediator, we performed microtubule regrowth assays under
conditions of mTOR inhibition. As shown on Fig. 3 addition
of rapamycin strongly impairs microtubule growth and
attachment of CLIP170 to microtubules ends.
In 2008 we also have continued our research on potential
involvement of rapamycin independent complex of mTOR,
mTORC2 in dendritogenesis and spine formation. RNA
interference mediated Rictor knockdown in developing rat
hippocampal neurons in culture resulted in the significant
reduction of the total dendritic length and complexity
of dendritic arbor as well as in changes of number and
morphology of dendritic spines. Furthermore, negative
effects of Rictor knockdown on dendritic arbor were
reversed by over expression of dominant negative form of
RhoA, strongly suggesting, that mTORC2 exerts its effect on
dendrites by controlling actin dynamics. Recently, we have
also shown that effects of Rictor knockdown can be reversed
by coexpresison of constitutively active Akt, another known
target of mTORC2.
Establishing a link between local protein translation
and physiological dendritic arbor development
To study the role of local protein translation in dendritic
arbor development, we have continued our studies on
the effects of knockdown of proteins of mRNA dendritic
transport machinery on dendritic arbor development. With
use of siRNA technology we targeted major components
of mRNA transport machinery such as β-actin zipcode
binding protein 1 (ZBP-1) and Staufens 1 and 2 in
hippocampal neurons. Indeed in all 3 cases knockdown
led to simplification of dendritic arbor that in case of ZBP-
1 was reversed by treatment with the actin polymerizing
drug – jasplakinolide, pointing to actin mRNA transport
and local translation being a major function of ZBP-1 during
dendritogenesis. However, it is worth stressing that our
bioinformatic screen performed in collaboration of Dr. Enrico
Tongiorgii from Trieste, has identified additional 8 mRNAs
encoded in rat genome that are potential targets for ZBP-1
and are expressed in neurons. Our current aim is to confirm
these predictions experimentally and investigate role of
those newly identified ZBP-1 targets during dendritogenesis
and dendritic spine development.
Recently, it has been shown that ZBP-1 function is
regulated by phosphorylation by Src kinase. That raised two
important questions i) are other mRNA binding proteins
involved in dendritic mRNA transport and translational
silencing regulated by phosporylation, ii) which other kinases
are involved in this process? To address these questions we
tested 19 selected proteins of ribonucleoprotein complex
64 Annual Report 2008
(RNP) for existence of potential “generic” phosphorylation
sites and for the probability of phosphorylation by selected
panel of kinases using Netphos2.0 and NetphosK software
(Blom et al., 1999, J. Mol. Biol., 294: 1351; Blom et al., 2004,
Proteomics, 4: 1633), respectively. To avoid artifacts due
to the usage of a single algorithm we repeated analysis
of potential phosphorylation sites for ZBP1, Staufen1 and
Staufen2 using Scansite 2.0 software (Obenauer et al., 2003,
NAR, 31: 3635). Indeed, most of the obtained results were
identical in both types of analysis. The performed analysis
revealed few regularities. First, that phosphorylation of
RNP proteins is a common event. Among analyzed kinases,
PKC, PKA and tyrosine kinases ubiquitously phosphorylate
proteins of RNPs. Other kinases are more selective, and
p38MAPK phosphorylating only two substrates is the most
spectacular example. Finally, we could distinguish proteins of
RNPs potentially undergoing very heavy phosphorylation by
several kinases (ZBP1, Satufen1, Pumilio) and those potentially
very poorly regulated (Translin, hnRNPA2). Consequently, we
performed experiments to confirm bioinformatic predictions
regarding ZBP1 and Staufens. Indeed we were able to show
phosphorylation of Staufen1 by Src kinase that has not been
reported so far. Since neither NetphosK nor Scansite2.0
contain consensus phosphorylation motifs for our favorite,
mTOR kinase in their libraries we used newly developed
software, Group Based Position software (GPS2.0; Xue et
al., 2008, Mol Cell Proteomics, 7: 1598) in order to test if RNP
proteins can be phosphorylated by this kinase. Results of GPS
analysis revealed that almost all analyzed proteins contained
at least one highly probable phosphorylation site for mTOR.
The only exceptions were Translin and hnRNPA2. FMRP
contained medium probability consensus phosphorylation
site. Similar results were obtained also for ERKs, another group
of kinases capable of direct control of translation machinery.
We next preliminarily confirmed our prediction that ZBP1
phosphorylation depends on mTOR activity using 2D
protein electrophoresis. Results of both, bioinformatics and
preliminary experiments suggest that mTOR and ERKs jointly
control translational mRNA competence and translation itself
and orchestrate local translational environment in neurons.
Characterization of both mTOR-regulated cellular
processes and local protein synthesis role in pathologies
of central nervous system
Our group is involved also in research projects aiming
on understanding role of mTOR in neuropathology during
development and aging. Together with several Polish
groups (Commissioned Grant of the Ministry of Science
and Higher Education), we aim to define mTOR targets that
are responsible for the progress of tuberous sclerosis – a
multiorgan disease that severely affects the brain. One of
the characteristic features of this disease is upregulation of
mTOR activity due to mutations in its inhibitors – hamartin
and tuberin (TSC1/2 complex). Among the hallmarks of the
TSC that are brain related, are hypertrophy of neuronal cells
and development of subependymal giant cell astrocytomas
(SEGA, 5-15% of cases). Indeed, silencing tuberin at the early
stage of neuron development (3-8 days in vitro) with short
interfering RNA resulted in an increase in neuron soma size.
We used this observation as a readout for shRNA screen for