Activation of S6K2 in lymphocytes by PMA+Ionomycin.

By Bonnie LarsenBy Bonnie Larsen

OutlineOutline• Background Information:Background Information:

– S6K1S6K1– S6K2S6K2– Immune System ReviewImmune System Review

• HypothesisHypothesis• Materials and MethodsMaterials and Methods• ResultsResults• Future ExperimentsFuture Experiments

S6K1S6K1• 70kD serine/threonine kinase found in the 70kD serine/threonine kinase found in the

cytoplasmcytoplasm• Phosphorylates S6 on the 40S ribosomal Phosphorylates S6 on the 40S ribosomal

subunitsubunit• Translational UpregulationTranslational Upregulation• High levels in growing or proliferating cellsHigh levels in growing or proliferating cells• KO mice were smaller, but still able to KO mice were smaller, but still able to

phosphorylate S6?phosphorylate S6?

S6K2S6K2

• 54kD ser/thr kinase homologous to S6K1 54kD ser/thr kinase homologous to S6K1 in the catalytic and regulatory domains.in the catalytic and regulatory domains.

• Predominantly found in the nucleus.Predominantly found in the nucleus.

• Phosphorylates S6 in vitro.Phosphorylates S6 in vitro.

• Overlapping and Discrete rolesOverlapping and Discrete roles

• Immune System: Rapamycin sensitiveImmune System: Rapamycin sensitive

What role does S6K2 play in What role does S6K2 play in the immune system?the immune system?

How can we study How can we study S6K2 in leukocytes?S6K2 in leukocytes?

AND

S6K1 in lymphocytesS6K1 in lymphocytes

• S6K1 levels high in T-cell lines.S6K1 levels high in T-cell lines.

• Sensitive to Rapamycin and WortmanninSensitive to Rapamycin and Wortmannin

• CD-5, CD-28, IL-2, TCR crosslinking, CD-5, CD-28, IL-2, TCR crosslinking, PMA+Ionomycin (P+I)PMA+Ionomycin (P+I)

• B-cell surface receptor ligationB-cell surface receptor ligation

S6K2 in lymphocytesS6K2 in lymphocytes

• Expressed at high levels in leukocytes Expressed at high levels in leukocytes and spleenand spleen

• Regulated by PI3K and mTORRegulated by PI3K and mTOR

• ????

Lymphocyte ReviewLymphocyte Review• B lymphocytes and T lymphocytesB lymphocytes and T lymphocytes• B lymphocytes express surface antibodies as B lymphocytes express surface antibodies as

well as secrete them.well as secrete them.• T lymphocytes express a T-cell Receptor (TCR) T lymphocytes express a T-cell Receptor (TCR)

complex and are activated by antigen complex and are activated by antigen presenting cells and TCR crosslinking.presenting cells and TCR crosslinking.

• Activated T lymphocytes synthesize multiple Activated T lymphocytes synthesize multiple proteins, proliferate and differentiate.proteins, proliferate and differentiate.

T-cell signalingT-cell signaling

IL-2 AutocrineSignaling

Differentiation

Proliferation

Protein Synthesis

TCR CrosslinkingCo-receptor

How can we visualize How can we visualize S6K2 activation in T S6K2 activation in T

lymphocytes?lymphocytes?• PMA+Ionomycin mechanism of actionPMA+Ionomycin mechanism of action

• By-pass TCR crosslinkingBy-pass TCR crosslinking

• If S6K2 activation occurs, it will If S6K2 activation occurs, it will phosphorylate S6.phosphorylate S6.

• We can measure changes in S6 We can measure changes in S6 phosphorylation to analyze S6K2 phosphorylation to analyze S6K2 activity.activity.

Experimental DesignExperimental Design

Time Coursed Assay0,5,10,15,20,25,30,60

minutesActivation with P+I

S6K2 vs. S6K1Activation

by P+I

Rapamycin Sensitivity:20minutes incubationwith Rapamycin prior

to P+I Activation

Activation:PMA

IonomycinPMA+Ionomycin

Kinase AssayPhosphorimaging

Experimental DesignExperimental Design-Kinase Assay-Kinase Assay

P I

Cell Lysis

J77/U937

Kinase Assay continued…Kinase Assay continued…

Anti-S6K1/S6K2 PAS

Beads Wash

Kinase Assay continued:Kinase Assay continued:GST-S6 & 32P-ATP SDS-PAGE and

Coomassie Blue

Kinase Assay continued:Kinase Assay continued:

X-Ray Photography

Expected ResultsExpected Results

ANDAND

What they can tell us…What they can tell us…

0

40000

80000

120000

160000

200000

240000

Control P I P+I

Ph

osp

ho

rim

ag

er U

nit

sPMA+Ionomycin activates S6K2 PMA+Ionomycin activates S6K2 but not PMA or Ionomycin alone.but not PMA or Ionomycin alone.

20-30min. activation time with P+I 20-30min. activation time with P+I yields significant S6K2 activity.yields significant S6K2 activity.

0

50000

100000

150000

200000

250000

0 5 10 15 20 25 30 60

P+I Activation Time

Ph

osp

ho

rim

ag

ing

Un

its

P+I activation of S6K1 vs. S6K2P+I activation of S6K1 vs. S6K2

0

4 0 0 0 0

8 0 0 0 0

12 0 0 0 0

16 0 0 0 0

2 0 0 0 0 0

2 4 0 0 0 0

S6K 2 C S6K 2 P +I S6K 1 C S6K 1 P +I

P+I activation of S6K2 is P+I activation of S6K2 is Rapamycin Sensitive.Rapamycin Sensitive.

0

4 0 0 0 0

8 0 0 0 0

12 0 0 0 0

16 0 0 0 0

2 0 0 0 0 0

2 4 0 0 0 0

C R apa+C R apa+P R apa+I R apa+P +I P +I

Simplified overview of

S6K2 regulation in lymphocytes:

GHH

?? ??

S6K2

S6

protein synthesis

Rho family G Proteins mTOR

PI3-K PKC

PKB

CD28

TCR

*S6K1 HAS BEEN

OMITTED FOR CLARITY.

IONOMYCIN

RAPAMYCIN

PMA

What knowledge has What knowledge has been gained?been gained?

Where do we go from Where do we go from here?here?

Now that we know what to Now that we know what to expect with S6 phosphorylation expect with S6 phosphorylation

by S6K2, we can…by S6K2, we can…

• Look for other downstream effectors using S6 Look for other downstream effectors using S6 phosphorylation as a control.phosphorylation as a control.

• Construct mutant cell lines and observe Construct mutant cell lines and observe differences in S6K2 activity: differences in S6K2 activity: – Can S6K2 still be activated for phosphorylation if we Can S6K2 still be activated for phosphorylation if we

disrupt certain other cellular factors? disrupt certain other cellular factors? – If we change certain amino acids in the primary If we change certain amino acids in the primary

structure of S6K2 is activity increased or decreased?structure of S6K2 is activity increased or decreased?

Future Experiments Continued…Future Experiments Continued…

• Analyze effects of S6 phosphorylation.Analyze effects of S6 phosphorylation.• Look at types and levels of protein synthesis.Look at types and levels of protein synthesis.• Complete our understanding of the pathway Complete our understanding of the pathway

involved in S6K2 regulation.involved in S6K2 regulation.• Analyze other ways of S6K2 activation:Analyze other ways of S6K2 activation:

– TCR crosslinkingTCR crosslinking– IL-2 autocrine signalingIL-2 autocrine signaling– Specific signal transduction pathways important in Specific signal transduction pathways important in

lymphocyte proliferation.lymphocyte proliferation.

AcknowledgementsAcknowledgements

• Dr. Lee-FrumanDr. Lee-Fruman• Deborah, Sharsti, Sopheap, Hemant and Deborah, Sharsti, Sopheap, Hemant and

DerekDerek• Howard Hughes Medical Research InstituteHoward Hughes Medical Research Institute