[Frontiers in Bioscience 1, d19-29 March 1, 1996]
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CAVEAT LECTOR



MICROINJECTION STRATEGIES FOR THE STUDY OF MITOGENIC SIGNALING IN MAMMALIAN CELLS

Ned J.C.Lamb, Cecile Gauthier-Rouviere and Anne Fernandez.

Cell Biology Unit, C R B M , CNRS-INSERM, 1919, Route de Mende, F-34033, Montpellier Cedex. France.

Received 15/12/95; Accepted 30/01/96; On-line 03/01/96

3.1 Microinjection of purified kinases and phosphatases to investigate the regulatory mechanisms involved in mitogenic activation.

Entry into G1 phase involves switching from quiescence (G0) to a proliferative state, with the induction of "immediate-early" genes. Stimulation of these early genes does not necessarily require protein synthesis, implying that appropriate regulatory factors are present before growth factor stimulation (i. e. in G0) (2,19). This is the case for SRF, which is present in quiescent cells, and after the inductive signal has been received, induces the expression of the early genes, such as c-fos. The rapidity of proliferative activation is due to the presence of such proteins even in G0 phase, whose regulation is by virtue of post-translational modifications, and predominantly by phosphorylation. The potential role of different protein kinases or phosphatases activated by growth factors in the subsequent activation of transcription factors, has been studied by microinjection of different purified enzymes. In the case of c-fos gene expression, two kinases were studied : casein kinase II (CKII) and Ca+2/phospholipid-dependent protein kinase (C-kinase) (15,20). Microinjection of either purified CKII or C-kinase resulted in the induction of c-fos in quiescent cells (with similar kinetics as serum induction). CKII is a kinase which is recruited to the nucleus at the G0/G1 transition. This enzyme has different nuclear targets, suggesting its implication in the final step of mitogenic signaling i.e. phosphorylation of transcription factors. Following CKII microinjection into quiescent cells, SRF was phosophorylated (15), and such phosphorylation was shown to slightly increase the DNA binding affinity of SRF and to markedly increase the rate of SRE-SRF exchange in vitro (21,22). Moreover, the possibility of coinjecting oligonucleotides corresponding to different protein binding DNA sequences together with protein kinases, has allowed identification of whether a promoter sequence is involved in the transcriptional activation by a specific kinase pathway. The co-injection of SRE oligonucleotide with CKII or C-kinase showed that activation at the SRE site (previously shown to be necessary for serum-induced c-fos expression) is also absolutely required for c-fos expression induced by CKII and C-kinase (table 2).

Table 2: Effect of oligonucleotide microinjection on the expression and down-regulation of c-fos induced by serum, CKII, C-kinase and ras microinjection
Inducer of c-FosTime effect observedno oligonucleotides+ SRE+ TRE/FAP
Serum addition90 min

5 hrs

+

-

-

-

-

+

CKII addition90 min

5 hrs

+

-

-

-

+

+

C-kinase microinjection90 min

5 hrs

+

-

-

-

-

-

Oncogenic Ras microinjection90 min

5 hrs

+

-

-

-

-

-

- : less than 5% of microinjected cells show c-fos expression

+ : 80 to 100% of microinjected cells show c-fos expression

Indeed, microinjection of SRE oligonucleotides with CKII or C-kinase totally inhibited c-fos expression clearly showing that positively acting factors activated by CKII and C-kinase must bind to the c-fos SRE to induce its expression. Immediately adjacent and 3’ to SRE in the c-fos promoter is a short sequence corresponding to c-fos AP1 binding site also called TPA Responsive Element (TRE). Displacement of TRE binding factors by microinjection of TRE oligonucleotides resulted in a delayed induction of c-fos by serum and markedly prolonged expression of c-fos induced by serum or CKII indicating that TRE could be involved in the down-regulation of c-fos expression (20). However, TRE microinjection blocked c-fos induction by C-kinase or by ras oncogenic protein, suggesting that the induction of c-fos by these two effectors utilizes a pathway more restrictive and different from the pathway of induction by SRF or serum.(20) (table 2). In a similar manner, microinjection of protein phosphatases was used to probe their role in the activation or down-regulation of specific mitogenic pathways. In particular, using cells transfected with a reporter LacZ gene under the control of a TRE (recognized by the transcription factor complex AP-1), Alberts et al. (23) have shown that microinjection of purified protein phosphatase type 2A (PP2A) enhanced b-galactosidase expression after serum stimulation, whereas microinjection of phosphatase type 1 (PP1) did not . This effect was correlated with the dephosphorylation of negative regulatory sites on c-Jun, in vitro (23). Inversely, microinjection of PP2A had no effect on cAMP-induced expression of a reporter gene carrying a cAMP Responsive Element (CRE), whereas PP1 injection abolished that expression (23). In these examples, microinjection shown different regulatory roles for the two major phosphatases, in expression control by distinct mitogenic pathways.

This approach allows to rapidly and transiently increase the level of a kinase or phosphatase and follow the effect of this elevation either on substrate phosphorylation or on a particular physiologic process such as c-fos gene expression, cell shape modification, etc.). By direct injection of purified enzymes, one can specifically study the end result of activating a particular enzyme and its downstream effectors (see summary in table 1).

3.2 Microinjection of peptides and proteins inhibitory to kinases and phosphatases.

A complementary approach involves microinjection of a specific inhibitory peptide against kinases or phosphatases. This can be applied to studying the role of several different protein kinases (C-kinase, A-kinase, CKII), or phosphatases such as PP1. It may prove useful in situations where no inhibitory antibody is available. These peptides are synthesized based on the sequence of an endogenous inhibitory protein (for example PKI is a synthetic peptide analogous in sequence to a 20 amino acid proteolytic cleavage fragment which retains a high inhibitory activity for A-kinase and can be modified to increase its stability in vivo (24)). The peptide can be synthesized by inserting a pseudosubstrate site in the regulatory domain from a given protein. this strategy has been used in synthesizing C-PKI, a synthetic peptide which acts as a potent C-kinase substrate antagonist (25)). These two peptides are suitable as probes in living cells. In particular, microinjection of the C-PKI peptide has been used to show the requirement of a C-kinase activity for ras-induced c-fos expression (12). A-kinase inhibition by PKI inside living cells resulted in marked effects on chromatin structure and cytoskeletal organisation (26) resembling those that accompany mitotic entry. Indeed, A-kinase appears to be a specific antagonist of many mitotic pathways (26,27). Such a global loss of A-kinase activity which prevents basic cell functions, prevents probing specific aspects of the cell metabolism. For example microinjection of PKI results in the exclusion of most transcription factors from the nucleus. Indeed, using microinjection of PKI, it was recently demonstrated that A-kinase activity is required in the process of active nuclear import of all proteins, including SRF (28). Another recent study addressing the role of protein phosphatases, used microinjection of a plasmid coding for a constitutively active form of inhibitor 1, a specific inhibitor of protein phosphatase type 1 (PP1), (29). These experiments proved that the transcription factor (CREB) that binds and activates the cAMP Response Element (CRE) is dephosphorylated on its A-kinase site by PP1, thereby limiting the transcriptional activity of CREB (29). This example combines the strategy of applying inhibitory peptides to specifically inactivate an enzyme pathway, and the use of expression plasmid microinjection, which allows the sustained overexpression of a peptide or a protein, as detailed below.

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